Anti P2X7 receptor antibodies and fragments thereof

ABSTRACT

The invention relates to an antigen binding site for binding to a P2X 7  receptor, the antigen binding site being defined by general formula 1:
 
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is the national phase under 35 U.S.C. 371 ofPCT/AU2010/001070, filed Aug. 20, 2010, which claims priority toAustralian Application No. 2009903928, filed Aug. 20, 2009.

REFERENCE TO SEQUENCE LISTING

This application refers to a Sequence Listing provided as a textdocument. The document is entitled “05220841.txt” (104,124 bytes createdJul. 20, 2012) and is hereby incorporated by reference in its entiretyherein.

FIELD OF THE INVENTION

The invention relates to purinergic receptors, to antibodies and relatedfragments thereof for binding to said receptors, to production of saidantibodies and fragments and to use of said antibodies and fragments forcancer detection and therapy.

BACKGROUND OF THE INVENTION

Reference to any prior art in the specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in Australia or any otherjurisdiction or that this prior art could reasonably be expected to beascertained, understood and regarded as relevant by a person skilled inthe art.

Purinergic (P2X) receptors are ATP-gated cation-selective channels. Eachreceptor is made up of three protein subunits or monomers. To date sevenseparate genes encoding P2X monomers have been identified: P2X1, P2X2,P2X3, P2X4, P2X5, P2X6, P2X₇.

P2X₇ receptors are of particular interest as the expression of thesereceptors is understood to be limited to cells having potential toundergo programmed cell death, such as thymocytes, dendritic cells,lymphocytes, macrophages and monocytes. There is some expression of P2X₇receptors in normal homeostasis, such as on erythrocytes.

Interestingly, a P2X₇ receptor containing one or more monomers having acis isomerisation at Pro210 (according to SEQ ID NO: 1) and which isdevoid of ATP binding function has been found on cells that areunderstood to be unable to undergo programmed cell death, such aspreneoplastic cells and neoplastic cells. This isoform of the receptorhas been referred to as a “non functional” receptor.

Antibodies generated from immunisation with a peptide including Pro210in cis bind to non functional P2X₇ receptors. However, they do not bindto P2X₇ receptors capable of binding ATP. Accordingly, these antibodiesare useful for selectively detecting many forms of carcinoma andhaemopoietic cancers and to treatment of some of these conditions.

WO02/057306A1 and WO03/020762A1 both discuss a probe for distinguishingbetween functional P2X₇ receptors and non functional P2X₇ receptors inthe form of a monoclonal antibody.

WO2009/033233 discusses an epitope present on non functional receptorsbut not functional receptors and antibodies for binding thereto.

To date it has been very difficult to obtain serological reagents thatbind to non functional P2X₇ receptors on live cells with desirableaffinity. Higher affinity reagents are generally desirable inapplications for the detection and treatment of cancer.

There is a need for improved reagents for binding to P2X₇ receptors,particularly for new antibodies and fragments thereof that are capableof discriminating between ATP and non-ATP binding P2X₇ receptors on livecells.

SUMMARY OF THE INVENTION

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 1:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR1 has a sequence selected from the group consisting of: DNEPMG (SEQID NO: 4), RNHDMG (SEQ ID NO: 7), SGYAMA (SEQ ID NO: 10), GMYNMS (SEQ IDNO: 13), PASNMS (SEQ ID NO: 16), GSYAMA (SEQ ID NO: 19), GAYAMS (SEQ IDNO: 22), DGYNMSSEQ ID NO: 25), TYDMAW (SEQ ID NO: 28), QEYGMG (SEQ IDNO: 31), ARYPMA (SEQ ID NO: 34), SSYAMA (SEQ ID NO: 37), AKYPMV (SEQ IDNO: 40), SSYAMS (SEQ ID NO: 43), DNVEMS (SEQ ID NO: 46) and PMKDMG (SEQID NO: 49).

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 2:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR2 has a sequence selected from the group consisting of:SIADSGNHTYYADSVKG (SEQ ID NO: 5), AISGSGGSTYYADSVKG (SEQ ID NO: 8),TILSDGSRTYYADSVKG (SEQ ID NO: 11), SINATGGRTYYADSVKG (SEQ ID NO: 14),SITASGYRTYYADSVKG (SEQ ID NO: 17), TISTSGSSTYYADSVKG (SEQ ID NO: 20),TINGSGLATYYADSVKG (SEQ ID NO: 23), SITANGNSTYYADSVKG (SEQ ID NO: 26),SIAAAGSRTYYADSVKG (SEQ ID NO: 29), SITPSGDKTYYADSVKG (SEQ ID NO: 32),SIDGGGLQTYYADSVKG (SEQ ID NO: 35), TIDGNGLITYYADSVKG (SEQ ID NO: 38),SIGPGGARTYYADSVKG (SEQ ID NO: 41), TITSDGLRTYYADSVKG (SEQ ID NO: 44),SIGSKGEDTYYADSVKG (SEQ ID NO: 47), AISGSGGSTYYANSVKG (SEQ ID NO: 53),AISGSGGGTYYADSVKG (SEQ ID NO: 110), SIGTKGEYTYYADSVKG (SEQ ID NO: 128),SIGSKGEYTYYADSVKG (SEQ ID NO: 131) and AISGSGGGTYYANSVKG (SEQ ID NO:137).

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 3:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has a sequence selected from the group consisting of:KQRGLNRYRAQFDY (SEQ ID NO: 6), EPKPMDTEFDY (SEQ ID NO: 9),KIKTFRNHSVQFDY (SEQ ID NO: 12), KFNGFSHRQYNFDY (SEQ ID NO: 15),KQGQISNFPRFDY (SEQ ID NO: 18), KVRFATSKSINFDY (SEQ ID NO: 21),KCSSCTSLNANFDY (SEQ ID NO: 24), KASYSRPYNFQFDY (SEQ ID NO: 27),KQRSISIRPMFDY (SEQ ID NO: 30), KVRSMSYAHFDFDY (SEQ ID NO: 33),KASAPKYFRFDY (SEQ ID NO: 36), KLQRYDRYTLNFDY (SEQ ID NO: 39),KPWRVYSYDRFDY (SEQ ID NO: 42), KVHTFANRSLNFDY (SEQ ID NO: 45),QTVNVPEPAFAY (SEQ ID NO: 48) and EPSHFDRPFDY (SEQ ID NO: 51).

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 4:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR1 has a sequence selected from the group consisting of:(P/R)(N/M)(H/K)DMG (SEQ ID NO: 199)

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 5:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR2 has a sequence selected from the group consisting of:AISGSGG(S/G)TYYA(D/N)SVKG (SEQ ID NO: 200).

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 6:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has a sequence selected from the group consisting of:EP(K/S)(P/H)(M/F)D(T/R)(E/P)FDY (SEQ ID NO: 201).

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 7:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has a sequence: EP(K/S)(P/H)(M/F)D(T/R)(E/P)FDY (SEQ ID NO: 201);andFR4 has a sequence: (W/R/P/G/C)(G/S/F)(Q/P/C)GT(L/Q)VTV(S/L)(S/E) (SEQID NO: 202).

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 8:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR1 has a sequence: (P/R)(N/M)(H/K)DMG (SEQ ID NO: 199);CDR2 has a sequence: AISGSGG(S/G)TYYA(D/N)SVKG (SEQ ID NO: 200);CDR3 has a sequence: EP(K/S)(P/H)(M/F)D(T/R)(E/P)FDY (SEQ ID NO: 201);andFR4 has a sequence: (W/R/P/G/C)(G/S/F)(Q/P/C)GT(L/Q)VTV(S/L)(S/E) (SEQID NO: 202).

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 9:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR1 has a sequence: (P/R)(N/M)(H/K)DMG (SEQ ID NO: 199);CDR2 has a sequence: AISGSGG(S/G)TYYA(D/N)SVKG (SEQ ID NO: 200);CDR3 has a sequence: EP(K/S)(P/H)(M/F)D(T/R)(E/P)FDY (SEQ ID NO: 201);FR1 has a sequence: EVQLLE(S/P)GGGLVQPGGSLRLSCAASG(Y/F/V)(R/T/N)(I/F/V)(SEQ ID NO: 203);FR2 has a sequence: W(V/A)RQAPGKGLEW(V/A)S (SEQ ID NO: 204);FR3 has a sequence: RFTISRDNS(R/K)NTLYLQMNS(L/M)RAEDTAVYYCA (SEQ ID NO:205);FR4 has a sequence: (W/R/P/G/C)(G/S/F)(Q/P/C)GT(L/Q)VTV(S/L)(S/E) (SEQID NO: 202).

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 10:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR1 has a sequence: PMKDMG (SEQ ID NO: 49);CDR2 has a sequence: AISGSGGGTYYADSVKG (SEQ ID NO: 110);CDR3 has a sequence: EPKPMDTEFDY (SEQ ID NO: 9);FR1 has a sequence: EVQLLESGGGLVQPGGSLRLSCAASGYTF (SEQ ID NO: 142);FR2 has a sequence: WVRQAPGKGLEWVS (SEQ ID NO: 145);FR3 has a sequence: RFTISRDNSKNTLYLQMNSLRAEDTAVYYCA (SEQ ID NO: 148);FR4 has a sequence: PSPGTLVTVLE (SEQ ID NO: 169), WGQGTLVTVSS (SEQ IDNO: 153), WGQGTLVTVLS (SEQ ID NO: 154), RSPGTLVTVSS (SEQ ID NO: 155),PSPGTQVTVSS (SEQ ID NO: 156), PSPGTLVTVSS (SEQ ID NO: 157), RSQGTLVTVSS(SEQ ID NO: 158), WSQGTLVTVSS (SEQ ID NO: 159), RGQGTLVTVSS (SEQ ID NO:160), RFQGTLVTVSS (SEQ ID NO: 161), WSPGTLVTVSS (SEQ ID NO: 162),GSPGTLVTVSS (SEQ ID NO: 163), WGPGTLVTVSS (SEQ ID NO: 164), RGPGTLVTVSS(SEQ ID NO: 165), CGPGTLVTVSS (SEQ ID NO: 166), RSCGTLVTVSS (SEQ ID NO:167), or RSPGTLVTVLE (SEQ ID NO: 168).

In other embodiments there is provided an antigen binding site having asequence as described herein, or including a CDR and/or FR sequence asdescribed herein and including one or more mutations for increasing theaffinity of said site for binding to a P2X₇ receptor.

In another embodiment there is provided an antigen binding site asdescribed herein wherein an amino acid sequence forming one or more ofFR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 is a human sequence.

In another embodiment there is provided an anti P2X₇ receptorimmunoglobulin variable domain, antibody, Fab, dab, scFv including anantigen binding site having a sequence as described herein, or includinga CDR and/or FR sequence as described herein.

In another embodiment there is provided a diabody or triabody includingan antigen binding site having a sequence as described herein, orincluding a CDR and/or FR sequence as described herein.

In another embodiment there is provided a fusion protein including anantigen binding site, immunoglobulin variable domain, antibody, Fab,dab, scFv, diabody or triabody as described herein.

In another embodiment there is provided a conjugate in the form of anantigen binding site, immunoglobulin variable domain, antibody, Fab,dab, scFv, diabody, triabody or fusion protein as described hereinconjugated to a label or a cytotoxic agent.

In another embodiment there is provided an antibody for binding to anantigen binding site of an immunoglobulin variable domain, antibody,Fab, dab, scFv, diabody, triabody, fusion protein, or conjugate asdescribed herein.

In another embodiment there is provided a nucleic acid encoding anantigen binding site, or a CDR and/or FR sequence as described herein,or an immunoglobulin variable domain, antibody, Fab, dab, scFv, diabody,triabody, fusion protein or conjugate as described herein.

In another embodiment there is provided a vector including a nucleicacid described herein.

In another embodiment there is provided a cell including a vector ornucleic acid described herein.

In another embodiment there is provided an animal or tissue derivedtherefrom including a cell described herein.

In another embodiment there is provided a pharmaceutical compositionincluding an antigen binding site, or including a CDR and/or FR sequenceas described herein, or an immunoglobulin variable domain, antibody,Fab, dab, scFv, diabody, triabody, fusion protein, or conjugate asdescribed herein and a pharmaceutically acceptable carrier, diluent orexcipient.

In another embodiment there is provided a diagnostic compositionincluding an antigen binding site, or including a CDR and/or FR sequenceas described herein, or an immunoglobulin variable domain, antibody,Fab, dab, scFv, diabody, triabody, fusion protein or conjugate asdescribed herein, a diluent and optionally a label.

In another embodiment there is provided a kit or article of manufactureincluding an antigen binding site, or including a CDR and/or FR sequenceas described herein or an immunoglobulin variable domain, antibody, Fab,dab, scFv, diabody, triabody, fusion protein or conjugate as describedherein.

In another embodiment there is provided a use of a sequence according toone or more of CDR1, CDR2, FR1, FR2, FR3 and FR4 as described herein toproduce an antigen binding site for binding to a P2X₇ receptor.

In another embodiment there is provided a use of an antigen binding siteor a CDR and/or FR sequence as described herein to produce an anti P2X₇receptor antigen binding site having increased affinity for P2X₇receptor.

In another embodiment there is provided a library of nucleic acidmolecules produced from the mutation of an antigen binding site or a CDRand/or FR sequence as described herein, wherein at least one nucleicacid molecule in said library encodes an antigen binding site forbinding to an a P2X₇ receptor.

In another embodiment there is provided a method for producing an antiP2X₇ antigen binding site as described herein including expressing anucleic acid as described herein in a cell or animal as describedherein.

In another embodiment there is provided a method for the treatment ofcancer or a condition or disease associated with expression of nonfunctional P2X₇ receptor in an individual including the step ofproviding an antigen binding site, immunoglobulin variable domain,antibody, Fab, dab, scFv, diabody, triabody, fusion protein, conjugateor pharmaceutical composition as described herein to an individualrequiring treatment for cancer or said condition or disease.

In another embodiment there is provided a use of an antigen bindingsite, immunoglobulin variable domain, antibody, Fab, dab, scFv, diabody,triabody, fusion protein, conjugate or pharmaceutical composition asdescribed herein in the manufacture of a medicament for the treatment ofcancer or a condition or disease associated with expression of nonfunctional P2X₇ receptor.

In another embodiment there is provided a method for the diagnosis ofcancer or disease or condition associated with expression of nonfunctional P2X₇ receptor, including the step of contacting tissues orcells for which the presence or absence of cancer is to be determinedwith a reagent in the form of an antigen binding site, immunoglobulinvariable domain, antibody, Fab, dab, scFv, diabody, triabody, fusionprotein, conjugate or diagnostic composition as described herein anddetecting for the binding of the reagent with the tissues or cells. Themethod may be operated in vivo or in vitro.

Typically the antigen binding sites according to the invention bind tonon functional P2X₇ receptors, especially receptors wherein Pro210 ofP2X₇ is in cis conformation. In certain embodiments the antigen bindingsites according to the invention do not bind to functional P2X₇receptors, especially receptors wherein Pro210 of P2X₇ is in transconformation.

Typically the antigen binding sites according to the invention bind tonon functional P2X₇ receptors on live cells. In other embodiments, theantigen binding site does not bind to receptors on dead or fixed cellstissues, such as those as studied in histology or cytology.

In one embodiment, the antigen binding sites according to the inventionbind to P2X₇ receptors on live cells with affinities in the range of 0.1to 5 nM.

In one embodiment, there is provided a single domain antibody includingan antigen binding site for binding to a P2X7 receptor, preferably to anon functional P2X7 receptor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Round 2 dAb ELISA positives screened on Biacore from the Round 2phage

FIG. 2. 20 nM PEP2-4, no peptide, cervical cancer, objective×10

FIG. 3. 20 nM PEP2-4, 0.1 mM peptide, cervical cancer, serial section,limited binding

FIG. 4. 20 nM PEP2-4, 1.0 mM peptide, cervical cancer, serial section,no binding

FIG. 5. 20 nM PEP2-4, no peptide, cervical cancer, objective×10

FIG. 6. 20 nM PEP2-4, 0.1 mM peptide, cervical cancer, serial section,limited binding

FIG. 7. 20 nM Pep2-4, 1.0 mM peptide, cervical cancer, serial section,no binding

FIG. 8. 20 nM Pep2-4, no peptide, cervical cancer

FIG. 9. 20 nM Pep2-4, 10 uM peptide, cervical cancer, serial section,binding unaffected

FIG. 10. 20 Nm PEP2-4 Melanoma, objective×20

FIG. 11. Lead dAb-Fc expressing at a molecular weight of 75 kDa

FIG. 12. Traces that can be easily resolved from the bottom include thecontrol dAb, HEL4-Fc (green), PEP2-47, PEP2-42, PEP2-42-1, PEP2-2 (blue)with other higher affinity binders above. Flow rate was 50 uL/min.

FIG. 13. The P2X₇ extracellular domain 47-332 with C-terminal c-Myc tagand N-terminal HA tag attached to PDGFR transmembrane anchorage for usein screening E200 conformational antigen binders expressed on HEK293cells.

FIG. 14. SDS-PAGE Western blot of cell lystate and surface expressedproteins expressing the ECD1, wild type (WT) P2X₇ and the twonon-functional P2X₇ receptor mutants R307Q and E496A. The ECD1 isexpressed at 52 kDa, the three P2X₇ receptors at 75 kDa. Anti-cadherincontrol in the lower section is at 98 kDa and anti-actin in the celllysate at 42 kDa.

FIG. 15. SDS-PAGE of ECD2 (47-306) and a mutant construct K193A(47-306)showing protein A fractions 1-5 and the supernatant (NB) with molecularweight standards at left.

FIG. 16. SDS-PAGE both non-reduced and reduced of dAb-Fc and ECD2-Fcalong with corresponding Western Blots reacted with anti-P2X₇ antibody.

FIG. 17. A selection of dAbs tested at 5 uM. Staining was detected withanti-human IgG Fab.

FIG. 18. Flow cytometry of the dAb-Fc binding to the pDisplay-ECD2 onHEK cells showing tighter cell binding by higher affinity species.

FIG. 19. Biacore tracings of selected PEP2-42 clones showing improvedbinding to E200 peptide.

FIG. 20. Sequences of PEP2-42 clones.

FIG. 21. Tree of affinity maturation pathways from lead binder toexpressed extracellular domain of target receptor

FIG. 22. Biacore traces of the PEP2-2-3 Fc clone at increasingconcentrations run against 10 RU of E200 peptide.

FIG. 23. Biacore traces of clones produced by NNS screening of Trp103 inPEP2-2-1.

FIG. 24. Binding by flow cytometry of PEP2-2-1 to cells expressing ECD1or ECD2 together with controls (mock and pDisplay only).

FIG. 25. Biacore tracings showing competitive binding between PEP2-2-1,E200 peptide and ECD2 (47-306).

FIG. 26. Lead dAbs 2-2-1 Fc, 2-2-3 Fc and 2-2-8 Fc binding to liveprostate PC3 cells at 0-20 ug/mL.

FIG. 27. Lead dAbs 2-2-1 Fc, 2-2-3 Fc and 2-2-8 Fc binding to livebreast MDA MB 231 cells at 0-20 ug/mL.

FIG. 28. Lead dAbs 2-2-1 Fc, 2-2-3 Fc and 2-2-8 Fc binding to liveovarian SKOV-3 cells at 0-20 ug/mL.

FIG. 29. Lead dAbs 2-2-1 Fc, 2-2-3 Fc and 2-2-8 Fc binding to live renal786-0 cells at 0-20 ug/mL.

FIG. 30. Lead dAbs 2-2-1 Fc, 2-2-3 Fc and 2-2-8 Fc binding to livemelanoma G361 cells at 0-20 ug/mL.

FIG. 31. Lead dAbs 2-2-1 Fc, 2-2-3 Fc and 2-2-8 Fc binding to live lungNCI-H596 cells at 0-20 ug/mL.

FIG. 32. Flow cytometry of human lymphocytes and monocytes from PBMCshowing no binding by PEP2-2-1 Fc or PEP2-2-3 Fc.

FIG. 33. Flow cytometry of prostate LNCap cells showing binding byPEP2-2-1 Fc, PEP2-2-3 Fc and HLA whereas the HEL4 control shows nobinding above the secondary alone.

FIG. 34. CTB assay showing inhibition of PC3 cell growth over 5 days inthe presence of increased PEP2-2-1 Fc and PEP2-2-3 Fc compared withcontrol HEL4 Fc.

FIG. 35. CTB assay showing inhibition of COLO205 cell growth over 3 and5 days in the presence of increased PEP2-2-1 Fc and PEP2-2-3 Fc comparedwith control HEL4 Fc.

FIG. 36. CTB assay showing inhibition of A375 cell growth over 3 and 5days in the presence of increased PEP2-2-1 Fc and PEP2-2-3 Fc comparedwith control HEL4 Fc

FIG. 37. Biacore traces of PEP2-2-12 dAb domain tested at 10, 5, 2.5, 1and 0.5 nM.

FIG. 38. Biacore traces of PEP2-2-12Alexa488 domain tested at 5 and 2.5nM.

FIG. 39. Biacore traces of PEP2-472-12Alexa488 domain tested at 10 and 5nM.

FIG. 40. Alignment of dAb sequences.

FIG. 41. (SEQ ID NO:1) Sequence of P2X₇.

FIG. 42. (SEQ ID NO:2) Sequence of ECD2

FIG. 43 (SEQ ID NO:3) Sequence of ECD1.

FIG. 44. Map of construct pcDNA3.1 PEP2-2-1 dAb-FC.

FIG. 45A-C (SEQ ID NO: 198) Sequence of pcDNA3.1 PEP2-2-1 dAb-FC.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Reference will now be made in detail to certain embodiments of theinvention. While the invention will be described in conjunction with theembodiments, it will be understood that the intention is not to limitthe invention to those embodiments. On the contrary, the invention isintended to cover all alternatives, modifications, and equivalents,which may be included within the scope of the present invention asdefined by the claims.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. The present invention is in no waylimited to the methods and materials described.

It will be understood that the invention disclosed and defined in thisspecification extends to all alternative combinations of two or more ofthe individual features mentioned or evident from the text or drawings.All of these different combinations constitute various alternativeaspects of the invention.

As used herein, except where the context requires otherwise, the term“comprise” and variations of the term, such as “comprising”, “comprises”and “comprised”, are not intended to exclude further additives,components, integers or steps.

For purposes of interpreting this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa. In the event thatany definition set forth conflicts with any document incorporated hereinby reference, the definition set forth below shall prevail.

“Purinergic receptor” generally refers to a receptor that uses a purine(such as ATP) as a ligand.

“P2X₇ receptor” generally refers to a purinergic receptor formed fromthree protein subunits or monomers, with at least one of the monomershaving an amino acid sequence substantially as shown in SEQ ID NO:1.“P2X₇ receptor” may be a functional or non functional receptor asdescribed below. “P2X₇ receptor” encompasses naturally occurringvariants of P2X₇ receptor, e.g., wherein the P2X₇ monomers are splicevariants, allelic variants and isoforms including naturally-occurringtruncated or secreted forms of the monomers forming the P2X₇ receptor(e.g., a form consisting of the extracellular domain sequence ortruncated form of it), naturally-occurring variant forms (e.g.,alternatively spliced forms) and naturally-occurring allelic variants.In certain embodiments of the invention, the native sequence P2X₇monomeric polypeptides disclosed herein are mature or full-length nativesequence polypeptides comprising the full-length amino acids sequenceshown in SEQ ID NO:1. In certain embodiments the P2X₇ receptor may havean amino acid sequence that is modified, for example various of theamino acids in the sequence shown in SEQ ID NO:1 may be substituted,deleted, or a residue may be inserted.

“Functional P2X₇ receptor” generally refers to a form of the P2X₇receptor having a binding site or cleft for binding to ATP. When boundto ATP, the receptor forms a pore-like structure that enables theingress of calcium ions into the cytosol, one consequence of which maybe programmed cell death. In normal homeostasis, expression offunctional P2X₇ receptors is generally limited to cells that undergoprogrammed cell death such as thymocytes, dendritic cells, lymphocytes,macrophages and monocytes. There may also be some expression offunctional P2X₇ receptors on erythrocytes.

“Non functional P2X₇ receptor” generally refers to a form of a P2X₇receptor in which one or more of the monomers has a cis isomerisation atPro210 (according to SEQ ID NO:1). The isomerisation may arise from anymolecular event that leads to misfolding of the monomer, including forexample, mutation of monomer primary sequence or abnormal posttranslational processing. One consequence of the isomerisation is thatthe receptor is unable to bind to ATP, or otherwise binds ATP with alower affinity than observed between ATP and receptors which do notcontain an isomerisation at Pro210. In the circumstances, the receptorcannot form a pore and this limits the extent to which calcium ions mayenter the cytosol. Non functional P2X₇ receptors are expressed on a widerange of epithelial and haematopoietic cancers.

“Extracellular domain” (ECD) used herein are P2X₇ receptor (47-306) (SEQID NO: 2) (ECD2) and P2X₇ receptor (47-332) (SEQ ID NO:3) (ECD1). P2X₇receptor (47-306) (SEQ ID NO: 2) is amino acids 47 to 306 of SEQ IDNO: 1. P2X₇ receptor (47-332) (SEQ ID NO:3) is amino acids 47 to 332 ofSEQ ID NO: 1.

“Antibodies” or “immunoglobulins” or “Igs” are gamma globulin proteinsthat are found in blood, or other bodily fluids of vertebrates thatfunction in the immune system to bind antigen, hence identifying andneutralizing foreign objects.

Antibodies are generally a heterotetrameric glycoprotein composed of twoidentical light (L) chains and two identical heavy (H) chains. Each Lchain is linked to a H chain by one covalent disulfide bond. The two Hchains are linked to each other by one or more disulfide bonds dependingon the H chain isotype. Each H and L chain also has regularly spacedintrachain disulfide bridges.

H and L chains define specific Ig domains. More particularly, each Hchain has at the N-terminus, a variable domain (V_(H)) followed by threeconstant domains (C_(H)) for each of the α and γ chains and four C_(H)domains for p and c isotypes. Each L chain has at the N-terminus, avariable domain (V_(L)) followed by a constant domain (C_(L)) at itsother end. The V_(L) is aligned with the V_(H) and the C_(L) is alignedwith the first constant domain of the heavy chain (C_(H)1).

Antibodies can be assigned to different classes or isotypes. There arefive classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, havingheavy chains designated α, δ, ε, γ, and μ, respectively. The γ and αclasses are further divided into subclasses on the basis of relativelyminor differences in C_(H) sequence and function, e.g., humans expressthe following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The Lchain from any vertebrate species can be assigned to one of two clearlydistinct types, called kappa and lambda, based on the amino acidsequences of their constant domains.

The constant domain includes the Fc portion which comprises thecarboxy-terminal portions of both H chains held together by disulfides.The effector functions of antibodies such as ADCC are determined bysequences in the Fc region, which region is also the part recognized byFc receptors (FcR) found on certain types of cells.

The pairing of a V_(H) and V_(L) together forms a “variable region” or“variable domain” including the amino-terminal domains of the heavy orlight chain of the antibody. The variable domain of the heavy chain maybe referred to as “VH.” The variable domain of the light chain may bereferred to as “VL.” The V domain contains an antigen binding site whichaffects antigen binding and defines specificity of a particular antibodyfor its particular antigen. V regions span about 110 amino acid residuesand consist of relatively invariant stretches called framework regions(FRs) (generally about 4) of 15-30 amino acids separated by shorterregions of extreme variability called “hypervariable regions” (generallyabout 3) that are each 9-12 amino acids long. The FRs largely adopt aβ-sheet configuration and the hypervariable regions form loopsconnecting, and in some cases forming part of, the β-sheet structure.

“Hypervariable region”, “HVR”, or “HV” refers to the regions of anantibody variable domain which are hypervariable in sequence and/or formstructurally defined loops. Generally, antibodies comprise sixhypervariable regions; three in the VH (HI, H2, H3), and three in the VL(LI, L2, L3). A number of hypervariable region delineations are in useand are encompassed herein. The Kabat Complementarity DeterminingRegions (CDRs) are based on sequence variability and are the mostcommonly used (Kabat et al, Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)).

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues herein defined.

“A peptide for forming an antigen binding site” generally refers to apeptide that may form a conformation that confers the specificity of anantibody for antigen. Examples include whole antibody or whole antibodyrelated structures, whole antibody fragments including a variabledomain, variable domains and fragments thereof, including light andheavy chains, or fragments of light and heavy chains that include somebut not all of hypervariable regions or constant regions.

An “intact” or “whole” antibody is one which comprises anantigen-binding site as well as a C_(L) and at least heavy chainconstant domains, C_(H)I, C_(H)2 and C_(H)3. The constant domains may benative sequence constant domains (e.g. human native sequence constantdomains) or amino acid sequence variant thereof.

“Whole antibody related structures” include multimerized forms of wholeantibody.

“Whole antibody fragments including a variable domain” include Fab,Fab′, F(ab′)₂, and Fv fragments; diabodies; linear antibodies,single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments.

The Fab fragment consists of an entire L chain along with the variableregion domain of the H chain (V_(H)), and the first constant domain ofone heavy chain (C_(H)I). Each Fab fragment is monovalent with respectto antigen binding, i.e., it has a single antigen-binding site.

A Fab′ fragment differs from Fab fragments by having additional fewresidues at the carboxy terminus of the C_(H)I domain including one ormore cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group.

A F(ab′)₂ fragment roughly corresponds to two disulfide linked Fabfragments having divalent antigen-binding activity and is still capableof cross-linking antigen.

An “Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association.

In a single-chain Fv (scFv) species, one heavy- and one light-chainvariable domain can be covalently linked by a flexible peptide linkersuch that the light and heavy chains can associate in a “dimeric”structure analogous to that in a two-chain Fv species. From the foldingof these two domains emanate six hypervariable loops (3 loops each fromthe H and L chain) that contribute the amino acid residues for antigenbinding and confer antigen binding specificity to the antibody.

“Single-chain Fv” also abbreviated as “sFV” or “scFV” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedto form a single polypeptide chain. Preferably, the scFv polypeptidefurther comprises a polypeptide linker between the V_(H) and V_(L)domains which enables the scFv to form the desired structure for antigenbinding.

A “single variable domain” is half of an Fv (comprising only three CDRsspecific for an antigen) that has the ability to recognize and bindantigen, although at a lower affinity than the entire binding site

“Diabodies” refers to antibody fragments with two antigen-binding sites,which fragments comprise a heavy-chain variable domain (VH) connected toa light-chain variable domain (VL) in the same polypeptide chain(V_(H)-V_(L)). The small antibody fragments are prepared by constructingsFv fragments (see preceding paragraph) with short linkers (about 5-10residues) between the V_(H) and V_(L) domains such that interchain butnot intra-chain pairing of the V domains is achieved, resulting in abivalent fragment, i.e., fragment having two antigen-binding sites.

Diabodies may be bivalent or bispecific. Bispecific diabodies areheterodimers of two “crossover” sFv fragments in which the V_(H) andV_(L) domains of the two antibodies are present on different polypeptidechains. Triabodies and tetrabodies are also generally know in the art.

An “isolated antibody” is one which has been identified and separatedand/or recovered from a component of its pre-existing environment.Contaminant components are materials that would interfere withtherapeutic uses for the antibody, and may include enzymes, hormones,and other proteinaceous or nonproteinaceous solutes.

A “human antibody” refers to an antibody which possesses an amino acidsequence which corresponds to that of an antibody produced by a humanand/or has been made using any of the techniques for making humanantibodies as disclosed herein. This definition of a human antibodyspecifically excludes a humanized antibody comprising non-humanantigen-binding residues. Human antibodies can be produced using varioustechniques known in the art, including phage-display libraries. Humanantibodies can be prepared by administering the antigen to a transgenicanimal that has been modified to produce such antibodies in response toantigenic challenge, but whose endogenous loci have been disabled.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies maycomprise residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

“Monoclonal antibody” refers to an antibody obtained from a populationof substantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. Monoclonalantibodies are highly specific, being directed against a singleantigenic site or determinant on the antigen. In addition to theirspecificity, the monoclonal antibodies are advantageous in that they maybe synthesized uncontaminated by other antibodies. Monoclonal antibodiesmay be prepared by the hybridoma methodology, or may be made usingrecombinant DNA methods in bacterial, eukaryotic animal or plant cells.The “monoclonal antibodies” may also be isolated from phage antibodylibraries.

The monoclonal antibodies herein include “chimeric” antibodies in whicha portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity. Chimeric antibodies of interest herein include“primatized” antibodies comprising variable domain antigen-bindingsequences derived from a non-human primate (e.g. Old World Monkey, Apeetc), and human constant region sequences.

The term “anti-P2X₇ receptor antibody” or “an antibody that binds toP2X₇ receptor” refers to an antibody that is capable of binding P2X₇receptor with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting P2X₇ receptor,typically non functional P2X₇ receptor. Preferably, the extent ofbinding of an P2X₇ receptor antibody to an unrelated receptor protein isless than about 10% of the binding of the antibody to P2X₇ receptor asmeasured, e.g., by a radioimmunoassay (RIA). In certain embodiments, anantibody that binds to P2X₇ receptor has a dissociation constant (Kd) of<1 μM, <100 nM, <10 nM, <1 nM, or <0.1 nM. An anti non functional P2X₇receptor antibody is generally one having some or all of theseserological characteristics and that binds to non functional P2X₇receptors but not to functional P2X₇ receptors.

An “affinity matured” antibody is one with one or more alterations inone or more HVRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). Preferred affinity matured antibodieswill have nanomolar or even picomolar affinities for the target antigen.Affinity matured antibodies are produced by procedures known in the art.

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Preferredblocking antibodies or antagonist antibodies substantially or completelyinhibit the biological activity of the antigen.

An “agonist antibody”, as used herein, is an antibody which mimics atleast one of the functional activities of a polypeptide of interest.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen).Generally, “binding affinity” refers to intrinsic binding affinity whichreflects a 1:1 interaction between members of a binding pair (e.g.,antibody and antigen). The affinity of a molecule X for its partner Ycan generally be represented by the dissociation constant (Kd). Affinitycan be measured by common methods known in the art, including thosedescribed herein. Low-affinity antibodies generally bind antigen slowlyand tend to dissociate readily, whereas high-affinity antibodiesgenerally bind antigen faster and tend to remain bound longer. A varietyof methods of measuring binding affinity are known in the art, any ofwhich can be used for purposes of the present invention.

The inventors have determined the CDR sequences of a number of variabledomain clones that they have found to bind to the ECD target. These CDRsequences are shown in Table 1 below.

In one embodiment there is provided a peptide having a sequence as shownin Table 1. These peptides are particularly useful for constructingantigen binding sites, variable domains, antibodies and relatedfragments.

TABLE 1 CDR sequences Clone CDR1 CDR2 CDR3 PEP2-1 SEQ ID NO: 4SEQ ID NO: 5 SEQ ID NO: 6 DNEPMG SIADSGNHTYYADSVKG KQRGLNRYRAQFDY PEP2-2SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 RNHDMG AISGSGGSTYYADSVKGEPKPMDTEFDY PEP2-3 SEQ ID NO: 10 SEQ ID NO: 11 SEQ ID NO: 12 SGYAMATILSDGSRTYYADSVKG KIKTFRNHSVQFDY PEP2-4 SEQ ID NO: 13 SEQ ID NO: 14SEQ ID NO: 15 GMYNMS SINATGGRTYYADSVKG KFNGFSHRQYNFDY PEP2-5SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: 18 PASNMS SITASGYRTYYADSVKGKQGQISNFPRFDY PEP2-6 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 21 GSYAMATISTSGSSTYYADSVKG KVRFATSKSINFDY PEP2-7 SEQ ID NO: 22 SEQ ID NO: 23SEQ ID NO: 24 GAYAMS TINGSGLATYYADSVKG KCSSCTSLNANFDY PEP2-8SEQ ID NO: 25 SEQ ID NO: 26 SEQ ID NO: 27 DGYNMS SITANGNSTYYADSVKGKASYSRPYNFQFDY PEP2-9 SEQ ID NO: 28 SEQ ID NO: 29 SEQ ID NO: 30 TYDMAWSIAAAGSRTYYADSVKG KQRSISIRPMFDY PEP2-10 SEQ ID NO: 31 SEQ ID NO: 32SEQ ID NO: 33 QEYGMG SITPSGDKTYYADSVKG KVRSMSYAHFDFDY PEP2-11SEQ ID NO: 34 SEQ ID NO: 35 SEQ ID NO: 36 ARYPMA SIDGGGLQTYYADSVKGKASAPKYFRFDY PEP2-13 SEQ ID NO: 37 SEQ ID NO: 38 SEQ ID NO: 39 SSYAMATIDGNGLITYYADSVKG KLQRYDRYTLNFDY PEP2-30 SEQ ID NO: 40 SEQ ID NO: 41SEQ ID NO: 42 AKYPMV SIGPGGARTYYADSVKG KPWRVYSYDRFDY PEP2-34SEQ ID NO: 43 SEQ ID NO: 44 SEQ ID NO: 45 SSYAMS TITSDGLRTYYADSVKGKVHTFANRSLNFDY PEP2-42 SEQ ID NO: 46 SEQ ID NO: 47 SEQ ID NO: 48 DNVEMSSIGSKGEDTYYADSVKG QTVNVPEPAFAY PEP2-47 SEQ ID NO: 49 SEQ ID NO: 50SEQ ID NO: 51 PMKDMG AISGSGGSTYYADSVKG EPSHFDRPFDY PEP2-2-1SEQ ID NO: 52 SEQ ID NO: 53 SEQ ID NO: 54 RNHDMG AISGSGGSTYYANSVKGEPKPMDTEFDY PEP2-2-1-1 SEQ ID NO: 55 SEQ ID NO: 56 SEQ ID NO: 57 RNHDMGAISGSGGSTYYADSVKG EPKPMDTEFDY PEP2-2-1-2 SEQ ID NO: 58 SEQ ID NO: 59SEQ ID NO: 60 RNHDMG AISGSGGSTYYADSVKG EPKPMDTEFDY PEP2-2-11SEQ ID NO: 61 SEQ ID NO: 62 SEQ ID NO: 63 RNHDMG AISGSGGSTYYANSVKGEPKPMDTEFDY PEP2-2-12 SEQ ID NO: 64 SEQ ID NO: 65 SEQ ID NO: 66 RNHDMGAISGSGGSTYYANSVKG EPKPMDTEFDY PEP2-2-2 SEQ ID NO: 67 SEQ ID NO: 68SEQ ID NO: 69 RNHDMG AISGSGGSTYYADSVKG EPKPMDTEFDY PEP2-2-4SEQ ID NO: 70 SEQ ID NO: 71 SEQ ID NO: 72 RNHDMG AISGSGGSTYYADSVKGEPKPMDTEFDY PEP2-2-5 SEQ ID NO: 73 SEQ ID NO: 74 SEQ ID NO: 75 RNHDMGAISGSGGSTYYADSVKG EPKPMDTEFDY PEP2-2-8 SEQ ID NO: 76 SEQ ID NO: 77SEQ ID NO: 78 RNHDMG AISGSGGSTYYADSVKG EPKPMDTEFDY PEP2-2-9SEQ ID NO: 79 SEQ ID NO: 80 SEQ ID NO: 81 RNHDMG AISGSGGSTYYADSVKGEPKPMDTEFDY PEP2-2-81 SEQ ID NO: 82 SEQ ID NO: 83 SEQ ID NO: 84 RNHDMGAISGSGGSTYYADSVKG EPKPMDTEFDY PEP2-2-91 SEQ ID NO: 85 SEQ ID NO: 86SEQ ID NO: 87 RNHDMG AISGSGGSTYYADSVKG EPKPMDTEFDY PEP2-2-3SEQ ID NO: 88 SEQ ID NO: 89 SEQ ID NO: 90 RNHDMG AISGSGGSTYYADSVKGEPKPMDTEFDY PEP2-2-31 SEQ ID NO: 91 SEQ ID NO: 92 SEQ ID NO: 93 RNHDMGAISGSGGSTYYADSVKG EPKPMDTEFDY PEP2-2-32 SEQ ID NO: 94 SEQ ID NO: 95SEQ ID NO: 96 RNHDMG AISGSGGSTYYADSVKG EPKPMDTEFDY PEP2-2-33SEQ ID NO: 97 SEQ ID NO: 98 SEQ ID NO: 99 RNHDMG AISGSGGSTYYADSVKGEPKPMDTEFDY PEP2-2-10 SEQ ID NO: 100 SEQ ID NO: 101 SEQ ID NO: 102RNHDMG AISGSGGSTYYADSVKG EPKPMDTEFDY PEP2-2-101 SEQ ID NO: 103SEQ ID NO: 104 SEQ ID NO: 105 RNHDMG AISGSGGSTYYADSVKG EPKPMDTEFDYPEP2-2-102 SEQ ID NO: 106 SEQ ID NO: 107 SEQ ID NO: 108 RNHDMGAISGSGGSTYYADSVKG EPKPMDTEFDY PEP2-247-1 SEQ ID NO: 109 SEQ ID NO: 110SEQ ID NO: 111 RNHDMG AISGSGGGTYYADSVKG EPSHFDRPFDY PEP2-247-2SEQ ID NO: 112 SEQ ID NO: 113 SEQ ID NO: 114 RNHDMG AISGSGGSTYYANSVKGEPSHFDRPFDY PEP2-472-1 SEQ ID NO: 115 SEQ ID NO: 116 SEQ ID NO: 117PMKDMG AISGSGGGTYYADSVKG EPKPMDTEFDY PEP2-472-11 SEQ ID NO: 118SEQ ID NO: 119 SEQ ID NO: 120 PMKDMG AISGSGGGTYYADSVKG EPKPMDTEFDYPEP2-472-12 SEQ ID NO: 121 SEQ ID NO: 122 SEQ ID NO: 123 PMKDMGAISGSGGGTYYADSVKG EPKPMDTEFDY PEP2-472-121 SEQ ID NO: 124 SEQ ID NO: 125SEQ ID NO: 126 PMKDMG AISGSGGGTYYADSVKG EPKPMDTEFDY PEP2-42-1SEQ ID NO: 127 SEQ ID NO: 128 SEQ ID NO: 129 DNVEMS SIGTKGEYTYYADSVKGQTVNVPEPAFAY PEP2-42-2 SEQ ID NO: 130 SEQ ID NO: 131 SEQ ID NO: 132DNVEMS SIGSKGEYTYYADSVKG QTVNVPEPAFAY PEP2-47-1 SEQ ID NO: 133SEQ ID NO: 134 SEQ ID NO: 135 PMKDMG AISGSGGGTYYADSVKG EPSHFDRPFDYPEP2-47-2 SEQ ID NO: 136 SEQ ID NO: 137 SEQ ID NO: 138 PMKDMGAISGSGGGTYYANSVKG EPSHFDRPFDY

The inventors have determined the FR sequences of a number of variabledomain clones that they have found to bind to the ECD target. These FRsequences are shown in Table 2 below. Other known FR sequences could beused with the above described CDRs to form an antigen binding site forbinding to a non functional P2X₇ receptor.

TABLE 2 Framework regions Clone FR1PEP2-1, PEP2-2, PEP2-3, PEP2-4, PEP2-5, PEP2-6, SEQ ID NO: 139PEP2-7, PEP2-8, PEP2-10, PEP2-11, PEP2-13, PEP2-EVQLLESGGGLVQPGGSLRLSCAASGFTF30, PEP2-34, PEP2-42, PEP2-47, PEP2-2-1, PEP2-2-1-1, PEP2-2-1-2, PEP2-2-11, PEP2-2-12, PEP2-2-2, PEP2-2-8, PEP2-2-9, PEP2-2-81, PEP2-2-91, PEP2-2-3, PEP2-2-31, PEP2-2-32, PEP2-2-33, PEP2-2-10, PEP2-2-101,PEP2-2-102, PEP2-247-1, PEP2-247-2, PEP2-42, PEP2- 42-1; PEP2-2-5 HEL-4SEQ ID NO: 140 EVQLLESGGGLVQPGGSLRLSCAASGFRI PEP2-9 SEQ ID NO: 141EVQLLESGGGLVQPGGSLRLSCAASGFTLPEP2-47-1, PEP2-47-2, PEP2-472-1, PEP2-472-11, SEQ ID NO: 142PEP2-472-12; PEP2-472-121 EVQLLESGGGLVQPGGSLRLSCAASGYTF PEP2-2-4SEQ ID NO: 143 EVQLLESGGGLVQPGGSLRLTCAASGFSF PEP2-42-2 SEQ ID NO: 144EVQMLESGGGLVQPGESLRLSCAASGFTF Clone FR2PEP2-1, PEP2-2, PEP2-4, PEP2-5, PEP2-6, PEP2-7, SEQ ID NO: 145PEP2-9, PEP2-10, PEP2-11, PEP2-13, PEP2-30, PEP2- WVRQAPGKGLEWVS34, PEP2-42, PEP2-47, PEP2-2-1, PEP2-2-1-1, PEP2-2-1-2, PEP2-2-11, PEP2-2-12, PEP2-2-2, PEP2-2-4, PEP2-2-5, PEP2-2-8, PEP2-2-9, PEP2-2-81, PEP2-2-91, PEP2-2-3, PEP2-2-31, PEP2-2-32, PEP2-2-33, PEP2-2-10,PEP2-2-101, PEP2-2-102, PEP2-472-1, PEP2-472-11,PEP2-472-12, PEP2-472-121, PEP2-247-1, PEP2-247-2,PEP2-42-1, PEP2-42-2, PEP2-47-1, PEP2-47-2 PEP2-3 SEQ ID NO: 146WVRQAPGKGLEWAS PEP2-8 SEQ ID NO: 147 WARQAPGKGLEWVS Clone FR3PEP2-1, PEP2-2, PEP2-3, PEP2-4, PEP2-5, PEP2-6, SEQ ID NO: 148PEP2-7, PEP2-8, PEP2-9, PEP2-10, PEP2-11, PEP2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYCA13, PEP2-30, PEP2-42, PEP2-47, PEP2-2-1, PEP2-2-1-1, PEP2-2-1-2, PEP2-2-11, PEP2-2-12, PEP2-2-2,PEP2-2-4, PEP2-2-8, PEP2-2-9, PEP2-2-81, PEP2-2-91, PEP2-2-3, PEP2-2-31, PEP2-2-32, PEP2-2-33,PEP2-2-10, PEP2-2-101, PEP2-2-102, PEP2-472-1,PEP2-472-11, PEP2-472-12, PEP2-472-121, PEP2-247-1, PEP2-247-2, PEP-2-47-1, PEP-2-47-2 PEP2-34 SEQ ID NO: 149RFTISRDNSRNTLYLQMNSLRAEDTAVYYCA PEP2-42-1 SEQ ID NO: 150RFTISRDNSKNTLYLQMNSMRAEDTAVYYCA PEP2-42-2 SEQ ID NO: 151RFTISRDNSKNTLYLQMNSPRAEDTAVYYCA PEP2-2-5 SEQ ID NO: 152RFTISRDDSKNTLYLQMNSLRAEDTAVYYCA Clone FR4PEP2-1, PEP2-2, PEP2-3, PEP2-4, PEP2-5, PEP2-6, SEQ ID NO: 153PEP2-7, PEP2-8, PEP2-9, PEP2-10, PEP2-11, PEP2-13, WGQGTLVTVSSPEP2-30, PEP2-34, PEP2-42, PEP2-47, PEP2-42-2 PEP2-42-1 SEQ ID NO: 154WGQGTLVTVLS PEP2-2-1, PEP2-2-1-1, PEP2-2-32, PEP2-2-4, PEP2-2-5SEQ ID NO: 155 RSPGTLVTVSS PEP2-2-11 SEQ ID NO: 156 PSPGTQVTVSSPEP2-2-12, PEP2-2-31 SEQ ID NO: 157 PSPGTLVTVSSPEP2-2-2, PEP2-47-1, PEP2-47-2, PEP2-472-1, PEP2- SEQ ID NO: 158247-1, PEP2-247-2 RSQGTLVTVSS PEP2-2-8, PEP2-2-81 SEQ ID NO: 159WSQGTLVTVSS PEP2-2-9, SEQ ID NO: 160 RGQGTLVTVSS PEP2-2-91SEQ ID NO: 161 RFQGTLVTVSS PEP2-2-3 SEQ ID NO: 162 WSPGTLVTVSS PEP2-2-33SEQ ID NO: 163 GSPGTLVTVSS PEP2-2-10 SEQ ID NO: 164 WGPGTLVTVSSPEP2-2-101 SEQ ID NO: 165 RGPGTLVTVSS PEP2-2-102 SEQ ID NO: 166CGPGTLVTVSS PEP2-472-11 SEQ ID NO: 167 RSCGTLVTVSS PEP2-472-12SEQ ID NO: 168 RSPGTLVTVLE PEP2-472-121 SEQ ID NO: 169 PSPGTLVTVLEPEP2-2-1-2 SEQ ID NO: 170 RSQGTLVTVSS

In certain embodiments there is provided an antigen binding site havinga sequence shown in Table 3 below:

TABLE 3 Antigen binding sites Clone Antigen binding site sequence PEP2-2SEQ ID NO: 171 PEP2-2EVQLLESGGGLVQPGGSLRLSCAASGFRIRNHDMGWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTE-FDYWGQGTLVTVSS PEP2-42 SEQ ID NO: 172 PEP2-42EVQLLESGGGLVQPGGSLRLSCAASGFTFDNVEMSWVRQAPGKGLEWVSSIGSKGEDTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAQTVNVPEPAFAYWGQGTLVTVSS PEP2-47 SEQ ID NO: 173 PEP2-47EVQLLESGGGLVQPGGSLRLSCAASGFTFPMKDMGWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPSHFDRP-FDYWGQGTLVTVSS PEP2-2- SEQ ID NO: 174 1 PEP2-2-1EVQLLESGGGLVQPGGSLRLSCAASGFTFRNHDMGWVRQAPGKGLEWVSAISGSGGSTYYANSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTE-FDYRSPGTLVTVSS PEP2-2- SEQ ID NO: 175 1-1 PEP2-2-1-1EVQLLESGGGLVQPGGSLRLSCAASGFTFRNHDMGWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTE-FDYRSPGTLVTVSS PEP2-2- SEQ ID NO: 176 1-2 PEP2-2-1-2EVQLLESGGGLVQPGGSLRLSCAASGFTFRNHDMGWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTE-FDYRSQGTLVTVSS PEP2-2- SEQ ID NO: 177 11 PEP2-2-11EVQLLESGGGLVQPGGSLRLSCAASGFTFRNHDMGWVRQAPGKGLEWVSAISGSGGSTYYANSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTE-FDYPSPGTQVTVSS PEP2-2- SEQ ID NO: 178 12 PEP2-2-12EVQLLESGGGLVQPGGSLRLSCAASGFTFRNHDMGWVRQAPGKGLEWVSAISGSGGSTYYANSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTE-FDYPSPGTLVTVSS PEP2-2- SEQ ID NO: 179 2 PEP2-2-2EVQLLESGGGLVQPGGSLRLSCAASGFTFRNHDMGWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTE-FDYRSQGTLVTVSS PEP2-2- SEQ ID NO: 180 4 PEP2-2-4EVQLLESGGGLVQPGGSLRLTCAASGFSFRNHDMGWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTE-FDYRSPGTLVTVSS PEP2-2- SEQ ID NO: 181 5 PEP2-2-5EVQLLESGGGLVQPGGSLRLSCAASGFTFRNHDMGWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTE-FDYRSPGTLVTVSS PEP2-2- SEQ ID NO: 182 8 PEP2-2-8EVQLLESGGGLVQPGGSLRLSCAASGFTFRNHDMGWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTE-FDYFSQGTLVTVSS PEP2-2- SEQ ID NO: 183 9 PEP2-2-9EVQLLESGGGLVQPGGSLRLSCAASGFTFRNHDMGWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTE-FDYRFQGTLVTVSS PEP2-2- SEQ ID NO: 184 3 PEP2-2-3EVQLLESGGGLVQPGGSLRLSCAASGFTFRNHDMGWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTE-FDYWSPGTLVTVSS PEP2-2- SEQ ID NO: 185 10 PEP2-2-10EVQLLESGGGLVQPGGSLRLSCAASGFTFRNHDMGWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTE-FDYRFPGTLVTVSS PEP2-2- SEQ ID NO: 186 101 PEP2-2-101EVQLLESGGGLVQPGGSLRLSCAASGFTFRNHDMGWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTE-FDYRGPGTLVTVSS PEP2-2- SEQ ID NO: 187 102 PEP2-2-102EVQLLESGGGLVQPGGSLRLSCAASGFTFRNHDMGWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTE-FDYCGPGTLVTVSS PEP2- SEQ ID NO: 188 472-1 PEP2-472-1EVQLLESGGGLVQPGGSLRLSCAASGYTFPMKDMGWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTE-FDYRSQGTLVTVSS PEP2- SEQ ID NO: 189 472-11 P2-472-11EVQLLESGGGLVQPGGSLRLSCAASGYTFPMKDMGWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTE-FDYRSCGTLVTVSS PEP2- SEQ ID NO: 190 472-12 P2-472-12EVQLLESGGGLVQPGGSLRLSCAASGYTFPMKDMGWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTE-FDYRSPGTLVTVSS PEP2- SEQ ID NO: 191 472-121 P2-472-121EVQLLESGGGLVQPGGSLRLSCAASGYTFPMKDMGWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTE-FDYPSPGTLVTVSS PEP2 SEQ ID NO: 192 247-1 PEP2-247-1EVQLLESGGGLVQPGGSLRLSCAASGFTFRNHDMGWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPSHFDRP-FDYRSQGTLVTVSS PEP2- SEQ ID NO: 193 247-2 PEP2-247-2EVQLLESGGGLVQPGGSLRLSCAASGFTFRNHDMGWVRQAPGKGLEWVSAISGSGGSTYYANSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPSHFDRP-FDYRSQGTLVTVSS PEP2- SEQ ID NO: 194 42-1 PEP2-42-1EVQMLESGGGLVQPGGSLRLSCAASGFTFDNVEMSWVRQAPGKGLEWVSSIGTKGEYTYYADSVKGRFTISRDNSKNTLYLQMNSMRAEDTAVYYCAQTVNVPEPAFAYWGQGTLVTVLS PEP2- SEQ ID NO: 195 42-2 PEP2-42-2EVQMLESGGGLVQPGESLRLSCAASGFTFDNVEMSWVRQAPGKGLEWVSSIGSKGEYTYYADSVKGRFTISRDNSKNTLYLQMNSPRAEDTAVYYCAQTVNVPEPAFAYWGQGTLVTVSS PEP2- SEQ ID NO: 196 47-1 PEP2-47-1EVQLLESGGGLVQPGGSLRLSCAASGYTFPMKDMGWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPSHFDRP-FDYRSQGTLVTVSS PEP2- SEQ ID NO: 197 47-2 PEP2-47-2EVQLLESGGGLVQPGGSLRLSCAASGYTFPMKDMGWVRQAPGKGLEWVSAISGSGGGTYYANSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPSHFDRP-FDYRSQGTLVTVSS

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 11:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR1 has a sequence selected from the group consisting of:(R/P/D)(N/M)(H/K/V)(D/E)M(G/S)

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 12:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR2 has a sequence selected from the group consisting of:(A/S)I(S/G)(G/S/T)(S/K)G(G/E)(S/G/D/Y)TYYA(D/N)SVKG (SEQ ID NO: 206).In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 13:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has a sequence selected from the group consisting of:(E/Q)(P/T)(K/S/V)(P/H/N)(M/F/V)(D/P)(T/R/E)(E/P)(A¹)F(A/D)Ywherein A¹ refers to no amino acid between (E/P) and F or alanine.

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 14:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has a sequence:(E/Q)(P/T)(K/S/V)(P/H/N)(M/F/V)(D/P)(T/R/E)(E/P)(A¹)F(A/D)YandFR4 has a sequence: (W/R/P/G/C)(G/S/F)(Q/C/P)GT(L/Q)VTV(S/L)(S/E) (SEQID NO: 202).

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 15:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR1 has a sequence: (R/P/D)(N/M)(H/K/V)(D/E)M(G/S);CDR2 has a sequence: (A/S)I(S/G)(G/S/T)(S/K)G(G/E)(S/G/D/Y)TYYA(D/N)SVKGSEQ ID NO: 206);CDR3 has a sequence:(E/Q)(P/T)(K/S/V)(P/H/N)(M/F/V)(D/P)(T/R/E)(E/P)(A¹)F(A/D)Y; wherein A¹refers to no amino acid between (E/P) and F or alanine.andFR4 has a sequence: (W/R/P/G/C)(G/S/F)(Q/C/P)GT(L/Q)VTV(S/L)(S/E) (SEQID NO: 202).

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 16:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR1 has a sequence: (R/P/D)(N/M)(H/K/V)(D/E)M(G/S);CDR2 has a sequence: (A/S)I(S/G)(G/S/T)(S/K)G(G/E)(S/G/D/Y)TYYA(D/N)SVKG(SEQ ID NO: 206);CDR3 has a sequence:(E/Q)(P/T)(K/S/V)(P/H/N)(M/F/V)(D/P)(T/R/E)(E/P)(A¹)F(A/D)Y, wherein A¹refers to no amino acid between (E/P) and F or alanine;FR1 has a sequence: EVQLLE(S/P)GGGLVQPGGSLRLSCAASG(Y/F/V)(R/T/N)(I/F/V)(SEQ ID NO: 203);FR2 has a sequence: WVRQAPGKGLEWVS (SEQ ID NO: 145);FR3 has a sequence: RFTISRDNSKNTLYLQMNS(L/M)RAEDTAVYYCA (SEQ ID NO:207);FR4 has a sequence: (W/R/P/G/C)(G/S/F)(Q/C/P)GT(L/Q)VTV(S/L)(S/E) (SEQID NO: 202).

In certain embodiments the antigen binding site is one having at least75%, preferably 80%, more preferably 85%, more preferably 90%, morepreferably 95%, more preferably 98% or 99% identity to an antigenbinding site shown in Table 3.

In certain embodiments the CDR is one having at least 75%, preferably80%, more preferably 85%, more preferably 90%, more preferably 95%, morepreferably 98% or 99% identity to a CDR shown in Table 1.

Percent sequence identity is determined by conventional methods, bymeans of computer programs known in the art such as GAP provided in theGCG program package (Program Manual for the Wisconsin Package, Version8, August 1994, Genetics Computer Group, 575 Science Drive, Madison,Wis., USA 53711) as disclosed in Needleman, S. B. and Wunsch, C. D.,(1970), Journal of Molecular Biology, 48, 443-453, which is herebyincorporated by reference in its entirety. GAP is used with thefollowing settings for polypeptide sequence comparison: GAP creationpenalty of 3.0 and GAP extension penalty of 0.1.

Sequence identity of polynucleotide molecules is determined by similarmethods using GAP with the following settings for DNA sequencecomparison: GAP creation penalty of 5.0 and GAP extension penalty of0.3.

In other embodiments there is provided an antigen binding site or CDRand/or FR having a sequence as described above and including one or moremutations for increasing the affinity of said site for binding to ananti-P2X₇ receptor. The mutation may result in a substitution, insertionor deletion of a residue in one or more of CDR1, CDR2 or CDR3, or one ormore or FR1, FR2, FR3 or FR4.

Marks et al. (1992) BioTechnology 10:779, which describes affinitymaturation by VH and VL domain shuffling; Barbas et al. (1994) Proc Nat.Acad. Sci. USA 9 1:3809; Schier et al. (1995) Gene 169:147-155; Yeltonet al. (1995) J. Immunol. 155:1994; Jackson et al (1995), J. Immunol.154(7):3310; and Hawkins et al, (1992) J. Mol. Biol. 226:889, whichdescribe random mutagenesis of hypervariable region and/or frameworkresidues, are examples of procedures known in the art for affinitymaturation of antigen binding sites. In certain embodiments, a nucleicacid encoding one or more of the sequences shown in Table 1 or Table 3is mutagenized to create a diverse library of sequences. The library isthen screened against a target including an epitope of a non functionalP2X₇ receptor. An exemplary method is shown in the Examples herein.

In another embodiment there is provided an antigen binding site asdescribed above wherein an amino acid sequence forming one or more ofFR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 is derived from a human sequenceor in the form of a human sequence.

The antigen binding site may be presented in a humanized form includingnon-human (e.g., murine) and human immunoglobulin sequences. Typicallyall but the CDR sequences of the antigen binding site are from anon-human species such as mouse, rat or rabbit. In some instances,framework residues of the antigen binding site may also be non human.Where the antigen binding site is provided in the form of a wholeantibody, typically at least a portion of an immunoglobulin constantregion (Fc) is human, thereby allowing various human effector functions.

Methods for humanizing non-human antigen binding sites are well known inthe art, examples of suitable processes including those in Jones et al.,(1986) Nature, 321:522; Riechmann et al., (1988) Nature, 332:323;Verhoeyen et al., (1988) Science, 239:1534.

Phage display methods described herein using antibody libraries derivedfrom human immunoglobulin sequences are useful for generating humanantigen binding sites and human antibodies.

Also, transgenic mammals that are incapable of expressing functionalendogenous immunoglobulins, but which can express human immunoglobulingenes can be used. These mice may be generated by random or targetedinsertion of the human heavy and light chain immunoglobulin genes intoembryonic stem cells. The host heavy and light chain immunoglobulingenes may be rendered non-functional by the insertion or by some otherrecombination event, for example by homozygous deletion of the host JHregion. The transfected embryonic stem cells are expanded andmicroinjected into blastocysts to produce chimeric mice that are thenbred to produce homozygous offspring that express human antigen bindingsites. After immunization with a P2X₇ epitope, human monoclonalantibodies can be obtained. One benefit of transgenic animal systems isthat it is possible to produce therapeutically useful isotypes becausethe human immunoglobulin transgenes rearrange during B-celldifferentiation and subsequently undergo class switching and somaticmutation in the transgenic mice.

Variable domains including CDRs and FRs of the invention may have beenmade less immunogenic by replacing surface-exposed residues so as tomake the antibody appear as self to the immune system. Padlan, E. A.,1991, Mol. Immunol. 28, 489 provides an exemplary method. Generally,affinity is preserved because the internal packing of amino acidresidues in the vicinity of the antigen binding site remains unchangedand generally CDR residues or adjacent residues which influence bindingcharacteristics are not to be substituted in these processes.

In another embodiment there is provided an anti P2X₇ receptorimmunoglobulin variable domain, antibody, Fab, dab or scFv including anantigen binding site as described above. In certain embodiments theantigen binding site has a sequence as shown in Table 3.

Lower molecular weight antibody fragments, as compared with wholeantibodies may have improved access to solid tumors and more rapidclearance which may be particularly useful in therapeutic and in vivodiagnostic applications.

Various techniques have been developed for the production of antibodyfragments including proteolytic digestion of intact antibodies andrecombinant expression in host cells. With regard to the latter, asdescribed below, Fab, Fv and scFv antibody fragments can all beexpressed in and secreted from E. coli, antibody fragments can beisolated from the antibody phage libraries and Fab′-SH fragments can bedirectly recovered from E. coli and chemically coupled to form F(ab′)2fragments. In another approach, F(ab′)2 fragments are isolated directlyfrom recombinant host cell culture.

In certain embodiments, the antigen binding site is provided in the formof a single chain Fv fragment (scFv). Fv and scFv are suitable forreduced nonspecific binding during in vivo use as they have intactcombining sites that are devoid of constant regions. Fusion proteinsincluding scFv may be constructed to yield fusion of an effector proteinat either the amino or the carboxy terminus of an scFv.

In another embodiment there is provided a diabody or triabody or othermultispecific antibody including an antigen binding site as describedabove. Multispecific antibodies may be assembled using polypeptidedomains that allow for multimerization. Examples include the CH2 and CH3regions of the Fc and the CH1 and Ckappa/lambda regions. Other naturallyoccurring protein multimerization domains may be used including leucinezipper domain (bZIP), helix-loop-helix motif, Src homology domain (SH2,SH3), an EF hand, a phosphotyrosine binding (PTB) domain, or otherdomains known in the art.

In another embodiment there is provided a fusion domain or heterologousprotein including an antigen binding site, immunoglobulin variabledomain, antibody, Fab, dab, scFv, diabody or triabody as describedabove.

A heterologous polypeptide may be recombinantly fused or chemicallyconjugated to an N- or C-terminus of an antigen binding site or moleculecontaining same of the invention.

The heterologous polypeptide to which the antibody or antigen bindingsite is fused may be useful to target to the P2X₇ receptor expressingcells, or useful to some other function such as purification, orincreasing the in vivo half life of the polypeptides, or for use inimmunoassays using methods known in the art.

In preferred embodiments, a marker amino acid sequence such as ahexa-histidine peptide (SEQ ID NO: 208) is useful for convenientpurification of the fusion protein. Others include, but are not limitedto, the “HA” tag, which corresponds to an epitope derived from theinfluenza hemagglutinin protein and the “flag” tag.

Further, the antigen binding site, immunoglobulin variable domain,antibody, Fab, dab, scFv, diabody or triabody of the invention may bemodified by glycosylation, acetylation, pegylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to a cellular ligand or other protein,etc.

Antigen binding sites of the invention can be composed of amino acidsjoined to each other by peptide bonds or modified peptide bonds, i.e.,peptide isosteres, and may contain amino acids other than the 20gene-encoded amino acids. Antigen binding sites of the invention may bemodified by natural processes, such as posttranslational processing, orby chemical modification techniques which are well known in the art.Such modifications are well described in basic texts, as well as inresearch literature. Modifications can occur anywhere in the antigenbinding site, including the peptide backbone, the amino acid side-chainsand the amino or carboxyl termini, or on moieties such as carbohydrates.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given antigenbinding site. Also, a given antigen binding site may contain many typesof modifications. An antigen binding site may be branched, for example,as a result of ubiquitination, and they may be cyclic, with or withoutbranching. Cyclic, branched, and branched cyclic antigen binding sitesmay result from posttranslation natural processes or may be made bysynthetic methods. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination.

In another embodiment there is provided a conjugate in the form of anantigen binding site, immunoglobulin variable domain, antibody, Fab,dab, scFsv, diabody, triabody or fusion protein as described aboveconjugated to a cytotoxic agent such as a chemo therapeutic agent, adrug, a growth inhibitory agent, a toxin (e.g., an enzymatically activetoxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a label such as a radioactive isotope (i.e., a radioconjugate). In another aspect, the invention further provides methods ofusing the immunoconjugates. In one aspect, an immunoconjugate comprisesany of the above variable domains covalently attached to a cytotoxicagent or a detectable agent.

In another embodiment there is provided an antibody for binding to anantigen binding site of an immunoglobulin variable domain, antibody,Fab, dab, scFv, diabody, triabody, fusion protein or conjugate asdescribed above.

In another embodiment there is provided a nucleic acid encoding anantigen binding site, immunoglobulin variable domain, antibody, Fab,dab, scFv, diabody, triabody, fusion protein or conjugate as describedabove.

A polynucleotide encoding an CDR or FR according to any one of thegeneral formulae described above, or an antigen binding site comprisedof same, may be generated from a nucleic acid from any source, forexample by chemical synthesis or isolation from a cDNA or genomiclibrary. For example a cDNA library may be generated from an antibodyproducing cell such as a B cell, plasma cell or hybridoma cell and therelevant nucleic acid isolated by PCR amplification usingoligonucleotides directed to the particular clone of interest. Isolatednucleic acids may then be cloned into vectors using any method known inthe art. The relevant nucleotide sequence may then be mutagenized usingmethods known in the art e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY), to generate antigen binding sites having a different aminoacid sequence, for example to create amino acid substitutions,deletions, and/or insertions.

In another embodiment there is provided a vector including a nucleicacid described above. The vector may, for example, be in the form of aplasmid, cosmid, viral particle, or phage. The appropriate nucleic acidsequence may be inserted into the vector by a variety of procedures. Ingeneral, DNA is inserted into an appropriate restriction endonucleasesite(s) using techniques known in the art. Vector components generallyinclude, but are not limited to, one or more of a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence. Construction ofsuitable vectors containing one or more of these components employsstandard ligation techniques which are known to the skilled artisan.

The antigen binding site may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which may be a signal sequence or other polypeptide havinga specific cleavage site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the antigen binding site-encoding DNAthat is inserted into the vector. The signal sequence may be aprokaryotic signal sequence selected, for example, from the group of thealkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin IIleaders. For yeast secretion the signal sequence may be, e.g., the yeastinvertase leader, alpha factor leader, or acid phosphatase leader or theC. albicans glucoamylase leader. In mammalian cell expression, mammaliansignal sequences may be used to direct secretion of the protein, such assignal sequences from secreted polypeptides of the same or relatedspecies, as well as viral secretory leaders.

Polynucleotide sequences encoding polypeptide components of the antigenbinding site of the invention can be obtained using standard recombinanttechniques as described above. Polynucleotides can be synthesized usingnucleotide synthesizer or PCR techniques. Once obtained, sequencesencoding the polypeptides are inserted into a recombinant vector capableof replicating and expressing heterologous polynucleotides inprokaryotic hosts. Many vectors that are available and known in the artcan be used for the purpose of the present invention. Selection of anappropriate vector will depend mainly on the size of the nucleic acidsto be inserted into the vector and the particular host cell to betransformed with the vector. Each vector contains various components,depending on its function (amplification or expression of heterologouspolynucleotide, or both) and its compatibility with the particular hostcell in which it resides.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. Both expression and cloning vectors containa nucleic acid sequence that enables the vector to replicate in one ormore selected host cells, as well as marking sequences which are capableof providing phenotypic selection in transformed cells. Such sequencesare well known for a variety of bacteria, yeast, and viruses. The originof replication from the plasmid pBR322, which contains genes encodingampicillin (Amp) and tetracycline (Tet) resistance and thus provideseasy means for identifying transformed cells, is suitable for mostGram-negative bacteria, the 2 μm plasmid origin is suitable for yeast,and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) areuseful for cloning vectors in mammalian cells. pBR322, its derivatives,or other microbial plasmids or bacteriophage may also contain, or bemodified to contain, promoters which can be used by the microbialorganism for expression of endogenous proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

The expression vector of the invention may comprise two or morepromoter-cistron (a cistron being segment of DNA that contains all theinformation for production of single polypeptide) pairs. A promoter isan untranslated regulatory sequence located upstream (5′) to a cistronthat modulates its expression. Prokaryotic promoters typically fall intotwo classes, inducible and constitutive. Inducible promoter is apromoter that initiates increased levels of transcription of the cistronunder its control in response to changes in the culture condition, e.g.the presence or absence of a nutrient or a change in temperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector of the invention. Both thenative promoter sequence and many heterologous promoters may be used todirect amplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters recognized by a variety of potential host cells are wellknown. Promoters suitable for use with prokaryotic hosts include thePhoA promoter, the β-galactamase and lactose promoter systems, alkalinephosphatase, a tryptophan (trp) promoter system and hybrid promoterssuch as the tac or the trc promoter. Promoters for use in bacterialsystems also will contain a Shine-Dalgarno (S.D.) sequence operablylinked to the DNA encoding an antigen binding site of the invention.However, other promoters that are functional in bacteria (such as otherknown bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledperson operably to ligate them to cistrons encoding the target light andheavy chains using linkers or adaptors to supply any requiredrestriction sites.

In one aspect of the invention, each cistron within the recombinantvector comprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. The signal sequence selected for the purpose of this inventionshould be one that is recognized and processed (i.e. cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the signal sequences native to the heterologouspolypeptides, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group consisting of thealkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II(STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of theinvention, the signal sequences used in both cistrons of the expressionsystem are STII signal sequences or variants thereof.

In another aspect, the production of the immunoglobulins according tothe invention can occur in the cytoplasm of the host cell, and thereforedoes not require the presence of secretion signal sequences within eachcistron. In that regard, immunoglobulin light and heavy chains areexpressed, folded and assembled to form functional immunoglobulinswithin the cytoplasm. Certain host strains (e.g., the E. coli trxB⁻strains) provide cytoplasm conditions that are favourable for disulfidebond formation, thereby permitting proper folding and assembly ofexpressed protein subunits.

The present invention provides an expression system in which thequantitative ratio of expressed polypeptide components can be modulatedin order to maximize the yield of secreted and properly assembledantigen binding sites of the invention. Such modulation is accomplishedat least in part by simultaneously modulating translational strengthsfor the polypeptide components.

In terms of expression in eukaryotic host cells, the vector componentsgenerally include, but are not limited to, one or more of the following:a signal sequence, an origin of replication, one or more marker genes,an enhancer element, a promoter, and a transcription terminationsequence.

A vector for use in a eukaryotic host cell may also contain a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide of interest. Theheterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

Generally, an origin of replication component is not needed formammalian expression vectors. For example, the SV40 origin may typicallybe used only because it contains the early promoter.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up the antigenbinding site-encoding nucleic acid, such as DHFR or thymidine kinase,metallothionein-I and -II, preferably primate metallothionein genes,adenosine deaminase, ornithine decarboxylase, etc. An appropriate hostcell when wild-type DHFR is employed is the CHO cell line deficient inDHFR activity (e.g., ATCC CRL-9096), prepared and propagated. Forexample, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR.Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418.

Expression and cloning vectors usually contain a promoter operablylinked to the antigen binding site encoding nucleic acid sequence todirect mRNA synthesis. Promoters recognized by a variety of potentialhost cells are well known.

Eukaryotic genes generally have an AT-rich region located approximately25 to 30 bases upstream from the site where transcription is initiated.Another sequence found 70 to 80 bases upstream from the start oftranscription of many genes is a CNCAAT region where N may be anynucleotide. At the 3′ end of most eukaryotic genes is an AATAAA sequencethat may be the signal for addition of the poly A tail to the 3′ end ofthe coding sequence. All of these sequences are suitably inserted intoeukaryotic expression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes including enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization.

Antigen binding site transcription from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40), from heterologous mammalian promoters, e.g., the actin promoteror an immunoglobulin promoter, and from heat-shock promoters, providedsuch promoters are compatible with the host cell systems.

Transcription of a DNA encoding the antigen binding site by highereukaryotes may be increased by inserting an enhancer sequence into thevector. Enhancer sequences include those known from mammalian genes(globin, elastase, albumin, α-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding an antigen binding site.

In another embodiment there is provided a cell including a vector ornucleic acid described above. The nucleic acid molecule or vector may bepresent in the genetically modified host cell or host either as anindependent molecule outside the genome, preferably as a molecule whichis capable of replication, or it may be stably integrated into thegenome of the host cell or host.

The host cell of the present invention may be any prokaryotic oreukaryotic cell.

Examples of prokaryotic cells are those generally used for cloning likeE. coli or Bacillus subtilis. Furthermore, eukaryotic cells comprise,for example, fungal or animal cells.

Examples for suitable fungal cells are yeast cells, preferably those ofthe genus Saccharomyces and most preferably those of the speciesSaccharomyces cerevisiae.

Examples of animal cells are, for instance, insect cells, vertebratecells, preferably mammalian cells, such as e.g. HEK293, NSO, CHO, MDCK,U2-OS, Hela, NIH3T3, MOLT-4, Jurkat, PC-12, PC-3, IMR, NT2N, Sk-n-sh,CaSki, C33A. These host cells, e.g. CHO-cells, may providepost-translational modifications to the antibody molecules of theinvention, including leader peptide removal, folding and assembly of H(heavy) and L (light) chains, glycosylation of the molecule at correctsides and secretion of the functional molecule.

Further suitable cell lines known in the art are obtainable from cellline depositories, like the American Type Culture Collection (ATCC).

In another embodiment there is provided an animal including a celldescribed above. In certain embodiments, animals and tissues thereofcontaining a transgene are useful in producing the antigen binding sitesof the invention. The introduction of the nucleic acid molecules astransgenes into non-human hosts and their subsequent expression may beemployed for the production of the antigen binding sites, for example,the expression of such a transgene in the milk of the transgenic animalprovide for means of obtaining the antigen binding sites in quantitativeamounts. Useful transgenes in this respect comprise the nucleic acidmolecules of the invention, for example, coding sequences for theantigen binding sites described herein, operatively linked to promoterand/or enhancer structures from a mammary gland specific gene, likecasein or beta-lactoglobulin. The animal may be non-human mammals, mostpreferably mice, rats, sheep, calves, dogs, monkeys or apes.

In another embodiment there is provided a pharmaceutical compositionincluding an antigen binding site, immunoglobulin variable domain,antibody, Fab, dab, scFv, diabody, triabody, fusion protein or conjugateas described above and a pharmaceutically acceptable carrier, diluent orexcipient.

Methods of preparing and administering antigen binding sites thereof toa subject in need thereof are well known to or are readily determined bythose skilled in the art. The route of administration of the antigenbinding site may be oral, parenteral, by inhalation or topical.

The term parenteral as used herein includes, e.g., intravenous,intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal orvaginal administration.

While all these forms of administration are clearly contemplated asbeing within the scope of the invention, a form for administration wouldbe a solution for injection, in particular for intravenous orintraarterial injection or drip. Usually, a suitable pharmaceuticalcomposition for injection may comprise a buffer (e.g. acetate, phosphateor citrate buffer), a surfactant (e.g. polysorbate), optionally astabilizer agent (e.g. human albumin), etc.

Preparations for parenteral administration includes sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. In the subject invention, pharmaceutically acceptable carriersinclude, but are not limited to, 0.01-0.1M and preferably 0.05Mphosphate buffer or 0.8% saline. Other common parenteral vehiclesinclude sodium phosphate solutions, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's, or fixed oils. Intravenous vehiclesinclude fluid and nutrient replenishers, electrolyte replenishers, suchas those based on Ringer's dextrose, and the like. Preservatives andother additives may also be present such as for example, antimicrobials,antioxidants, chelating agents, and inert gases and the like.

More particularly, pharmaceutical compositions suitable for injectableuse include sterile aqueous solutions (where water soluble) ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions, in such cases, thecomposition must be sterile and should be fluid to the extent that easysyringability exists. It should be stable under the conditions ofmanufacture and storage and will preferably be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Suitableformulations for use in the therapeutic methods disclosed herein aredescribed in Remington's Pharmaceutical Sciences, Mack Publishing Co.,16th ed. (1980).

Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating an activecompound (e.g., antigen binding site) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedherein, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle, which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying,which yields a powder of an active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The preparations for injections are processed, filled into containerssuch as ampoules, bags, bottles, syringes or vials, and sealed underaseptic conditions according to methods known in the art. Further, thepreparations may be packaged and sold in the form of a kit. Sucharticles of manufacture will preferably have labels or package insertsindicating that the associated compositions are useful for treating asubject suffering from, or predisposed disorders.

Effective doses of the compositions of the present invention, fortreatment of disorders as described herein vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human butnon-human mammals including transgenic mammals can also be treated.Treatment dosages may be titrated using routine methods known to thoseof skill in the art to optimize safety and efficacy.

For treatment of certain disorders with an antigen binding site, thedosage can range, e.g., from about 0.0001 to 100 mg/kg, and more usually0.01 to 5 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1mg/kg, 2 mg/kg, etc.), of the host body weight. For example dosages canbe 1 mg/kg body weight or 10 mg/kg body weight or within the range of1-10 mg/kg, preferably at least 1 mg/kg. Doses intermediate in the aboveranges are also intended to be within the scope of the invention.Subjects can be administered such doses daily, on alternative days,weekly or according to any other schedule determined by empiricalanalysis. An exemplary treatment entails administration in multipledosages over a prolonged period, for example, of at least six months.Additional exemplary treatment regimes entail administration once perevery two weeks or once a month or once every 3 to 6 months. Exemplarydosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30mg/kg on alternate days or 60 mg/kg weekly. In some methods, two or moreantigen binding sites with different binding specificities areadministered simultaneously, in which case the dosage of each antigenbinding sites administered falls within the ranges indicated.

An antigen binding site disclosed herein can be administered on multipleoccasions. Intervals between single dosages can be weekly, monthly oryearly. Intervals can also be irregular as indicated by measuring bloodlevels of target polypeptide or target molecule in the patient. In somemethods, dosage is adjusted to achieve a plasma polypeptideconcentration of 1-1000 μg/ml and in some methods 25-300 μg/ml.Alternatively, antigen binding sites can be administered as a sustainedrelease formulation, in which case less frequent administration isrequired. Dosage and frequency vary depending on the half-life of theantigen binding site in the patient. The half-life of an antigen bindingsite can also be prolonged via fusion to a stable polypeptide or moiety,e.g., albumin or PEG. In general, humanized antibodies show the longesthalf-life, followed by chimeric antibodies and nonhuman antibodies. Inone embodiment, the antigen binding site of the invention can beadministered in unconjugated form. In another embodiment the antigenbinding sites for use in the methods disclosed herein can beadministered multiple times in conjugated form. In still anotherembodiment, the antigen binding sites of the invention can beadministered in unconjugated form, then in conjugated form, or viceversa.

The dosage and frequency of administration can vary depending on whetherthe treatment is prophylactic or therapeutic. In prophylacticapplications, compositions comprising antibodies or a cocktail thereofare administered to a patient not already in the disease state or in apre-disease state to enhance the patient's resistance. Such an amount isdefined to be a “prophylactic effective dose.” In this use, the preciseamounts again depend upon the patient's state of health and generalimmunity, but generally range from 0.1 to 25 mg per dose, especially 0.5to 2.5 mg per dose. A relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives.

In therapeutic applications, a relatively high dosage (e.g., from about1 to 400 mg/kg of binding molecule, e.g., antigen binding site per dose,with dosages of from 5 to 25 mg being more commonly used forradioimmunoconjugates and higher doses for cytotoxin-drug conjugatedmolecules) at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the patient shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patent can be administered a prophylacticregime.

In one embodiment, a subject can be treated with a nucleic acid moleculeencoding an antigen binding site (e.g., in a vector). Doses for nucleicacids encoding polypeptides range from about 10 ng to 1 g, 100 ng to 100mg, 1 μg to 10 mg, or 30-300 μg DNA per patient. Doses for infectiousviral vectors vary from 10-100, or more, virions per dose. Therapeuticagents can be administered by parenteral, topical, intravenous, oral,subcutaneous, intraarterial, intracranial, intraperitoneal, intranasalor intramuscular means for prophylactic and/or therapeutic treatment, insome methods, agents are injected directly into a particular tissuewhere non-functional P2X₇ receptor cells have accumulated, for exampleintracranial injection. Intramuscular injection or intravenous infusionare preferred for administration of antibody, in some methods,particular therapeutic antibodies are injected directly into thecranium, in some methods, antibodies are administered as a sustainedrelease composition or device.

An antigen binding site of the invention can optionally be administeredin combination with other agents that are effective in treating thedisorder or condition in need of treatment (e.g., prophylactic ortherapeutic).

In another embodiment there is provided a pharmaceutical compositionincluding an antigen binding site, immunoglobulin variable domain,antibody, Fab, dab, scFv, diabody, triabody, fusion protein or conjugateas described above, a diluent and optionally a label.

In certain embodiments, the antigen binding sites or molecule includingsame are detectably labelled. Many different labels can be usedincluding enzymes, radioisotopes, colloidal metals, fluorescentcompounds, chemiluminescent compounds, and bioluminescent compounds.Fluorochromes (fluorescein, rhodamine, Texas Red, etc.), enzymes (horseradish peroxidase, β-galactosidase, alkaline phosphatase etc.),radioactive isotopes (³²P or ¹²⁵I), biotin, digoxygenin, colloidalmetals, chemi- or bioluminescent compounds (dioxetanes, luminol oracridiniums) are commonly used.

Detection methods depend on the type of label used and includeautoradiography, fluorescence microscopy, direct and indirect enzymaticreactions. Examples include Westernblotting, overlay-assays, RIA(Radioimmuno Assay) and IRMA (Immune Radioimmunometric Assay), EIA(Enzyme Immuno Assay), ELISA (Enzyme Linked Immuno Sorbent Assay), FIA(Fluorescent Immuno Assay), and CLIA (Chemioluminescent Immune Assay).

In another embodiment there is provided a kit or article of manufactureincluding an antigen binding site, immunoglobulin variable domain,antibody, Fab, dab, scFv, diabody, triabody, fusion protein, conjugateor pharmaceutical composition as described above.

In other embodiments there is provided a kit for use in a therapeuticapplication mentioned above, the kit including:

-   -   a container holding a therapeutic composition in the form of one        or more of an antigen binding site, immunoglobulin variable        domain, antibody, Fab, dab, scFv, diabody, triabody, fusion        protein, conjugate or pharmaceutical composition;    -   a label or package insert with instructions for use.

In certain embodiments the kit may contain one or more further activeprinciples or ingredients for treatment of a cancer or for preventing acancer-related complication described above, or a condition or diseaseassociated with non functional P2X₇ receptor expression.

The kit or “article of manufacture” may comprise a container and a labelor package insert on or associated with the container. Suitablecontainers include, for example, bottles, vials, syringes, blister pack,etc. The containers may be formed from a variety of materials such asglass or plastic. The container holds a therapeutic composition which iseffective for treating the condition and may have a sterile access port(for example the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). The labelor package insert indicates that the therapeutic composition is used fortreating the condition of choice. In one embodiment, the label orpackage insert includes instructions for use and indicates that thetherapeutic composition can be used to treat a cancer or to prevent acomplication stemming from cancer.

The kit may comprise (a) a therapeutic composition; and (b) a secondcontainer with a second active principle or ingredient containedtherein. The kit in this embodiment of the invention may furthercomprise a package insert indicating that the and other active principlecan be used to treat a disorder or prevent a complication stemming fromcancer. Alternatively, or additionally, the kit may further comprise asecond (or third) container comprising a pharmaceutically-acceptablebuffer, such as bacteriostatic water for injection (BWFI),phosphate-buffered saline, Ringer's solution and dextrose solution. Itmay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles, andsyringes.

In certain embodiments the therapeutic composition may be provided inthe form of a device, disposable or reusable, including a receptacle forholding the therapeutic composition. In one embodiment, the device is asyringe. The device may hold 1-2 mL of the therapeutic composition. Thetherapeutic composition may be provided in the device in a state that isready for use or in a state requiring mixing or addition of furthercomponents.

In another embodiment there is provided a kit or article of manufactureincluding an antigen binding site, immunoglobulin variable domain,antibody, Fab, dab, scFv, diabody, triabody, fusion protein, conjugateor a diagnostic composition as described above.

In other embodiments there is provided a kit for use in a diagnosticapplication mentioned above, the kit including:

-   -   a container holding a diagnostic composition in the form of one        or more of an antigen binding site, immunoglobulin variable        domain, antibody, Fab, dab, scFv, diabody, triabody, fusion        protein or conjugate;    -   a label or package insert with instructions for use.

The kit or “article of manufacture” may comprise a container and a labelor package insert on or associated with the container. Suitablecontainers include, for example, bottles, vials, syringes, blister pack,etc. The containers may be formed from a variety of materials such asglass or plastic. The container holds a diagnostic composition which iseffective for detection of cancer and may have a sterile access port(for example the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). The labelor package insert indicates that the diagnostic composition is used fordetecting the condition of choice. In one embodiment, the label orpackage insert includes instructions for use and indicates that thediagnostic composition can be used to detect a cancer or a disease orcondition characterised by non functional P2X₇ receptor expression.

The kit may comprise (a) a diagnostic composition; and (b) a secondcontainer with a second diagnostic agent or second label containedtherein. It may further include other materials desirable from acommercial and user standpoint, including other buffers, diluents,filters etc.

In another embodiment there is provided a method for producing an antiP2X₇ antigen binding site as described above including expressing anucleic acid as described above in a cell or non human animal asdescribed above.

The production of an antigen binding site of the invention generallyrequires an expression vector containing a polynucleotide that encodesthe antigen binding site of the invention. A polynucleotide encoding anantigen binding site of the invention may be obtained and sub clonedinto a vector for the production of an antigen binding site byrecombinant DNA technology using techniques well-known in the art,including techniques described herein. Many different expression systemsare contemplated including the use of mammalian cells including humancells for production and secretion of antigen binding sites. Examples ofcells include 293F, CHO and the NSO cell line.

Expression vectors containing protein coding sequences and appropriatetranscriptional and translational control signals can be constructedusing methods known in the art. These include in vitro recombinant DNAtechniques, synthetic techniques and in vivo genetic recombination. Incertain embodiments there is provided a replicable vector having anucleic acid encoding an antigen binding site operably linked to apromoter. Cells transfected with an expression vector may be cultured byconventional techniques to produce an antigen binding site. Thus, incertain embodiments, there is provided host cells or cell transfectantscontaining a polynucleotide encoding an antigen binding site of theinvention operably linked to a promoter. The promoter may beheterologous. A variety of host-expression vector systems may beutilized and in certain systems the transcription machinery of thevector system is particularly matched to the host cell. For example,mammalian cells such as Chinese hamster ovary cells (CHO) may betransfected with a vector including the major intermediate early genepromoter element from human cytomegalovirus. Additionally oralternatively, a host cell may be used that modulates the expression ofinserted sequences, or modifies and processes the gene product asrequired, including various forms of post translational modification.Examples of mammalian host cells having particular post translationmodification processes include CHO, VERY, BHK, Hela, COS, MDCK, 293,3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO, CRL7O3O and HsS78Bstcells.

Depending upon the use intended for the protein molecule, a number ofbacterial expression vectors may be advantageously selected. In oneexample, vectors that cause the expression of high levels of fusionprotein products that are readily purified, such as the E. coliexpression vector pUR278 may be used where a large quantity of anantigen binding site is to be produced. The expression product may beproduced in the form of a fusion protein with lacZ. Other bacterialvectors include pIN vectors and the like. pGEX vectors may also be usedto express foreign polypeptides as fusion proteins withglutathione-S-transferase (GST). These fusion proteins are generallysoluble and can easily be purified from lysed cells by adsorption andbinding to glutathione-agarose affinity matrix followed by elution inthe presence of free glutathione. A thrombin and/or factor Xa proteasecleavage site may be provided in the expressed polypeptide so that thecloned target gene product can be released from the GST moiety.

Autographa californica nuclear polyhedrosis virus (AcNPV) may be used asa vector to express foreign genes in an insect system includingSpodoptera frugiperda cells. The particular promoter used may depend onwhere the protein coding is inserted into the sequence. For example, thesequence may be cloned individually into the polyhedrin gene and placedunder control of the polyhedrin promoter.

Virus based expression systems may be utilized with mammalian cells suchas an adenovirus whereby the coding sequence of interest may be ligatedto the adenoviral late promoter and tripartite leader sequence. In vitroor in vivo recombination may then be used to insert this chimeric geneinto the adenoviral genome. Insertions into region E1 or E3 will resultin a viable recombinant virus that is capable of expressing the antigenbinding site in infected host cells. Specific initiation signalsincluding the ATG initiation codon and adjacent sequences may berequired for efficient translation of inserted antigen binding sitecoding sequences. Initiation and translational control signals andcodons can be obtained from a variety of origins, both natural andsynthetic. Transcription enhancer elements and transcription terminatorsmay be used to enhance the efficiency of expression of a viral basedsystem.

Where long-term, high-yield production of recombinant proteins isrequired, stable expression is preferred. Generally a selectable markergene is used whereby following transfection, cells are grown for 1-2days in an enriched media and then transferred to a medium containing aselective medium in which cells containing the corresponding selectablemarker, for example, antibiotic resistance can be screened. The resultis that cells that have stably integrated the plasmid into theirchromosomes grow and form foci that in turn can be cloned and expandedinto cell lines. The herpes simplex virus thymidine kinase,hypoxanthineguanine phosphoribosyltransferase and adeninephosphoribosyltransferase genes are examples of genes that can beemployed in tk⁻, hgprf⁻ or aprT⁻ cells, respectively thereby providingappropriate selection systems. The following genes: dhfr, which confersresistance to methotrexate; gpt, which confers resistance tomycophenolic acid; neo, which confers resistance to the aminoglycosideG-418; and hygro, which confers resistance to hygromycin are examples ofgenes that can be used in anti metabolite selection systems.

An antigen binding site of the invention may be purified by arecombinant expression system by known methods including ion exchangechromatography, affinity chromatography (especially affinity for thespecific antigens Protein A or Protein G) and gel filtration columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Purificationmay be facilitated by providing the antigen binding site in the form ofa fusion protein.

Large quantities of the antigen binding sites of the invention may beproduced by a scalable process starting with a pilot expression systemin a research laboratory that is scaled up to an analytical scalebioreactor (typically from 5 L to about 50 L bioreactors) or productionscale bioreactors (for example, but not limited to 75 L, 100 L, 150 L,300 L, or 500 L). Desirable scalable processes include those whereinthere are low to undetectable levels of aggregation as measured by HPSECor rCGE, typically no more than 5% aggregation by weight of protein downto no more than 0.5% by weight aggregation of protein. Additionally oralternatively, undetectable levels of fragmentation measured in terms ofthe total peak area representing the intact antigen binding site may bedesired in a scalable process so that at least 80% and as much as 99.5%or higher of the total peak area represents intact antigen binding site.In other embodiments, the scalable process of the invention producesantigen binding sites at production efficiency of about from 10 mg/L toabout 300 mg/L or higher.

In another embodiment there is provided a method for the treatment of adisease or condition characterised by non functional P2X₇ receptorexpression in an individual including the step of providing an antigenbinding site, immunoglobulin variable domain, antibody, Fab, dab, scFv,diabody, triabody, fusion protein, conjugate or pharmaceuticalcomposition as described above to an individual requiring treatment forsaid condition. Typically the condition is cancer, especially anepithelial cancer as described herein.

Pre-neoplastic and neoplastic diseases are particular examples to whichthe methods of the invention may be applied. Broad examples includebreast tumors, colorectal tumors, adenocarcinomas, mesothelioma, bladdertumors, prostate tumors, germ cell tumor, hepatoma/cholongio, carcinoma,neuroendocrine tumors, pituitary neoplasm, small 20 round cell tumor,squamous cell cancer, melanoma, atypical fibroxanthoma, seminomas,nonseminomas, stromal leydig cell tumors, Sertoli cell tumors, skintumors, kidney tumors, testicular tumors, brain tumors, ovarian tumors,stomach tumors, oral tumors, bladder tumors, bone tumors, cervicaltumors, esophageal tumors, laryngeal tumors, liver tumors, lung tumors,vaginal tumors and Wilm's tumor.

Examples of particular cancers include but are not limited toadenocarcinoma, adenoma, adenofibroma, adenolymphoma, adontoma, AIDSrelated. cancers, acoustic neuroma, acute lymphocytic leukemia, acutemyeloid leukemia, adenocystic carcinoma, adrenocortical cancer,agnogenic myeloid metaplasia, alopecia, alveolar soft-part sarcoma,ameloblastoma, angiokeratoma, angiolymphoid hyperplasia witheosinophilia, angioma sclerosing, angiomatosis, apudoma, anal cancer,angiosarcoma, aplastic anaemia, astrocytoma, ataxia-telangiectasia,basal cell carcinoma (skin), bladder cancer, bone cancers, bowel cancer,brain stem glioma, brain and CNS tumors, breast cancer, branchioma, CNStumors, carcinoid tumors, cervical cancer, childhood brain tumors,childhood cancer, childhood leukemia, childhood soft tissue sarcoma,chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronicmyeloid leukemia, colorectal cancers, cutaneous T-cell lymphoma,carcinoma (e.g. Walker, basal cell, basosquamous, Brown-Pearce, ductal,Ehrlich tumor, Krebs 2, Merkel cell, mucinous, non-small cell lung, oatcell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell,and transitional cell), carcinosarcoma, cervical dysplasia, cystosarcomaphyllodies, cementoma, chordoma, choristoma, chondrosarcoma,chondroblastoma, craniopharyngioma, cholangioma, cholesteatoma,cylindroma, cystadenocarcinoma, cystadenoma,dermatofibrosarcoma-protuberans, desmoplastic-small-round-cell-tumor,ductal carcinoma, dysgerminoam, endocrine cancers, endometrial cancer,ependymoma, esophageal cancer, Ewing's sarcoma, extra-hepatic bile ductcancer, eye cancer, eye: melanoma, retinoblastoma, fallopian tubecancer, fanconi anaemia, fibroma, fibrosarcoma, gall bladder cancer,gastric cancer, gastrointestinal cancers,gastrointestinal-carcinoid-tumor, genitourinary cancers, germ celltumors, gestationaltrophoblastic-disease, glioma, gynaecologicalcancers, giant cell tumors, ganglioneuroma, glioma, glomangioma,granulosa cell tumor, gynandroblastoma, haematological malignancies,hairy cell leukemia, head and neck cancer, hepatocellular cancer,hereditary breast cancer, histiocytosis, Hodgkin's disease, humanpapillomavirus, hydatidiform mole, hypercalcemia, hypopharynx cancer,hamartoma, hemangioendothelioma, hemangioma, hemangiopericytoma,hemangiosarcoma, hemangiosarcoma, histiocytic disorders, histiocytosismalignant, histiocytoma, hepatoma, hidradenoma, hondrosarcoma,immunoproliferative small, opoma, ontraocular melanoma, islet cellcancer, Kaposi's sarcoma, kidney cancer, langerhan's cell-histiocytosis,laryngeal cancer, leiomyosarcoma, leukemia, li-fraumeni syndrome, lipcancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma,Hodgkin's lymphoma, non-Hodgkin's lymphoma, leigomyosarcoma, leukemia(e.g. b-cell, mixed cell, null-cell, t-cell, t-cell chronic,htiv-ii-associated, lymphangiosarcoma, lymphocytic acute, lymphocyticchronic, mast-cell and myeloid), leukosarcoma, leydig cell tumor,liposarcoma, leiomyoma, leiomyosarcoma, lymphangioma, lymphangiocytoma,lymphagioma, lymphagiomyoma, lymphangiosarcoma, male breast cancer,malignant-rhabdoid-tumor-of-kidney, medulloblastoma, melanoma, Merkelcell cancer, mesothelioma, metastatic cancer, mouth cancer, multipleendocrine neoplasia, mycosis fungoides, myelodysplastic syndromes,myeloma, myeloproliferative disorders, malignant carcinoid syndromecarcinoid heart disease, medulloblastoma, meningioma, melanoma,mesenchymoma, mesonephroma, mesothelioma, myoblastoma, myoma,myosarcoma, myxoma, myxosarcoma, nasal cancer, nasopharyngeal cancer,nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegen breakagesyndrome, non-melanoma skin cancer, non-small-cell-lung-cancer-(nsclc),neurilemmoma, neuroblastoma, neuroepithelioma, neurofibromatosis,neurofibroma, neuroma, neoplasms (e.g. bone, breast, digestive system,colorectal, liver), ocular cancers, oesophageal cancer, oral cavitycancer, oropharynx cancer, osteosarcoma, ostomy ovarian cancer, pancreascancer, paranasal cancer, parathyroid cancer, parotid gland cancer,penile cancer, peripheral-neuroectodermal-tumors, pituitary cancer,polycythemia vera, prostate cancer, osteoma, osteosarcoma, ovariancarcinoma, papilloma, paraganglioma, paraganglioma nonchromaffin,pinealoma, plasmacytoma, protooncogene,rare-cancers-and-associated-disorders, renal cell carcinoma,retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson syndrome,reticuloendotheliosis, rhabdomyoma, salivary gland cancer, sarcoma,schwannoma, Sezary syndrome, skin cancer, small cell lung cancer (sclc),small intestine cancer, soft tissue sarcoma, spinal cord tumors,squamous-cell-carcinoma-(skin), stomach cancer, synovial sarcoma,sarcoma (e.g. Ewing's experimental, Kaposi's and mast-cell sarcomas),Sertoli cell tumor, synovioma, testicular cancer, thymus cancer, thyroidcancer, transitional-cell-cancer-(bladder),transitional-cell-cancer-(renal-pelvis-/-ureter), trophoblastic cancer,teratoma, theca cell tumor, thymoma, trophoblastic tumor, urethralcancer, urinary system cancer, uroplakins, uterine sarcoma, uteruscancer, vaginal cancer, vulva cancer, Waldenstrom's-macroglobulinemiaand Wilms' tumor.

Other diseases and conditions include various inflammatory conditions.Examples may include a proliferative component. Particular examplesinclude acne, angina, arthritis, aspiration pneumonia, disease, empyema,gastroenteritis, inflammation, intestinal flu, nee, necrotizingenterocolitis, pelvic inflammatory disease, pharyngitis, pid, pleurisy,raw throat, redness, rubor, sore throat, stomach flu and urinary tractinfections, chronic inflammatory demyelinating polyneuropathy, chronicinflammatory demyelinating polyradiculoneuropathy, chronic inflammatorydemyelinating polyneuropathy or chronic inflammatory demyelinatingpolyradiculoneuropathy.

In another embodiment there is provided a use of an antigen bindingsite, immunoglobulin variable domain, antibody, Fab, dab, scFsv,diabody, triabody, fusion protein, conjugate or pharmaceuticalcomposition as described above in the manufacture of a medicament forthe treatment of cancer.

Dosage amount, dosage frequency, routes of administration etc aredescribed in detail above.

In another embodiment there is provided a method for the diagnosis ofcancer including the step of contacting tissues or cells for which thepresence or absence of cancer is to be determined with a reagent in theform of an antigen binding site, immunoglobulin variable domain,antibody, Fab, dab, scFv, diabody, triabody, fusion protein, conjugateor diagnostic composition as described above and detecting for thebinding of the reagent with the tissues or cells. The method may beoperated in vivo or in vitro.

For in situ diagnosis, the antigen binding site may be administered tothe organism to be diagnosed by intravenous, intranasal,intraperitoneal, intracerebral, intraarterial injection or other routessuch that a specific binding between an antigen binding site accordingto the invention with an epitopic region on the non-functional P2X₇receptor may occur. The antibody/antigen complex may conveniently bedetected through a label attached to the antigen binding site or afunctional fragment thereof or any other art-known method of detection.

The immunoassays used in diagnostic applications according to theinvention and as described herein typically rely on labelled antigens,antibodies, or secondary reagents for detection. These proteins orreagents can be labelled with compounds generally known to those ofordinary skill in the art including enzymes, radioisotopes, andfluorescent, luminescent and chromogenic substances including, but notlimited to coloured particles, such as colloidal gold and latex beads.Of these, radioactive labelling can be used for almost all types ofassays and with most variations. Enzyme-conjugated labels areparticularly useful when radioactivity must be avoided or when quickresults are needed. Fluorochromes, although requiring expensiveequipment for their use, provide a very sensitive method of detection.Antibodies useful in these assays include monoclonal antibodies,polyclonal antibodies, and affinity purified polyclonal antibodies.

Alternatively, the antigen binding site may be labelled indirectly byreaction with labelled substances that have an affinity forimmunoglobulin, such as protein A or G or second antibodies. The antigenbinding site may be conjugated with a second substance and detected witha labelled third substance having an affinity for the second substanceconjugated to the antigen binding site. For example, the antigen bindingsite may be conjugated to biotin and the antigen binding site-biotinconjugate detected using labelled avidin or streptavidin. Similarly, theantigen binding site may be conjugated to a hapten and the antigenbinding site-hapten conjugate detected using labelled anti-haptenantibody.

In certain embodiments, immunoassays utilize a double antibody methodfor detecting the presence of an analyte, wherein, the antigen bindingsite is labelled indirectly by reactivity with a second antibody thathas been labelled with a detectable label. The second antibody ispreferably one that binds to antibodies of the animal from which theantigen binding site is derived. In other words, if the antigen bindingsite is a mouse antibody, then the labelled, second antibody is ananti-mouse antibody. For the antigen binding site to be used in theassay described herein, this label is preferably an antibody-coatedbead, particularly a magnetic bead. For the antigen binding site to beemployed in the immunoassay described herein, the label is preferably adetectable molecule such as a radioactive, fluorescent or anelectrochemiluminescent substance.

An alternative double antibody system, often referred to as fast formatsystems because they are adapted to rapid determinations of the presenceof an analyte, may also be employed within the scope of the presentinvention. The system requires high affinity between the antigen bindingsite and the analyte. According to one embodiment of the presentinvention, the presence of the non-functional P2X₇ receptor isdetermined using a pair of antigen binding sites, each specific for P2X₇receptor protein. One of said pairs of antigen binding sites is referredto herein as a “detector antigen binding site” and the other of saidpair of antigen binding sites is referred to herein as a “captureantigen binding site”. The antigen binding site of the present inventioncan be used as either a capture antigen binding site or a detectorantigen binding site. The antigen binding site of the present inventioncan also be used as both capture and detector antigen binding site,together in a single assay. One embodiment of the present invention thususes the double antigen binding site sandwich method for detectingnon-functional P2X₇ receptor in a sample of biological fluid. In thismethod, the analyte (non-functional P2X₇ receptor protein) is sandwichedbetween the detector antigen binding site and the capture antigenbinding site, the capture antigen binding site being irreversiblyimmobilized onto a solid support. The detector antigen binding sitewould contain a detectable label, in order to identify the presence ofthe antigen binding site-analyte sandwich and thus the presence of theanalyte.

Exemplary solid phase substances include, but are not limited to,microtiter plates, test tubes of polystyrene, magnetic, plastic or glassbeads and slides which are well known in the field of radioimmunoassayand enzyme immunoassay. Methods for coupling antigen binding sites tosolid phases are also well known to those of ordinary skill in the art.More recently, a number of porous material such as nylon,nitrocellulose, cellulose acetate, glass fibers and other porouspolymers have been employed as solid supports.

The examples that follow are intended to illustrate but in no way limitthe present invention.

EXAMPLES Example 1 Identifying Dab Leads for Binding to Non FunctionalReceptors on Live Cells

Objective:

The experiments described here have been to find antigen binding sitesthat bind the E200 peptide.

Background:

Antisera that bind P2X₇ have low affinity for P2X₇ as expressed on livecancer cells since the conformation of the epitope target on live cancercells differs. To identify dAb leads for high affinity binders, we firstneeded to identify a suitable target, knowing that good sequencediversity of binders is required in order to widen the screening ofconformational space to encompass suitable lead compounds. We selectedthe E200 peptide as a suitable target to identify dAb leads.

Materials and Methods:

The E200 peptide was made by solid phase synthesis at Chiron Mimotopes.A range of conjugates were synthesized to identify those most likely tobe useful for screening purposes. These included protein conjugates BSA,DT and ovalbumin linked to the C-terminal Cys reside on E200 peptide viaMCS. A fourth variant involved biotinylating the E200 peptide at theC-terminus.

Suitable lead clones were initially identified as ELISA positives inboth solid phase and solution phase screens. These were made againstboth the unconjugated and the conjugated peptides. Additional peptideswere synthesized (200-208 and 207-215) in order to differentiate morecompletely the binding regions of the various lead clones. Solutionproperties using SEC-MALLS of the lead clones were tested to ensure theywere suitable for further development.

Results:

A large number of first generation leads were identified and isolatedthat initially bound to the E200 peptide with binding affinity in the uMK_(D) range as measured by Biacore and then bound detectably by flowcytometry to live cancer cells expressing the non-functional P2X₇receptor target on their surface. Single domain antibodies produced fromDomantis phage display library screened against the peptide antigen E200exhibited a K_(D) of the order of 1 uM using Biacore binding analyses.Lead clones taken forward showed diversity in their bindingcharacteristics. Three lead dAbs, PEP2-2, PEP2-4 and PEP2-5 exhibitedthe highest affinity when tested on live PC3 human prostate cancer cellsby flow cytometry. Additional screening involved the use of standardimmunohistochemistry in which normal human and cancer tissue wasincubated with the chosen dAb labelled with Myc tag to which a labelledanti-Myc antibody with HRP was added. Diaminobenzoate (DAB) was added toreact with any HRP remaining after due washing steps were completed.PEP2-4 and PEP2-5 bound moderately to the tumour tissue but not tonormal tissue such as human prostate and skin while PEP2-2 was anexample of a dAb lead that showed little effective binding to tissue inthe initial screening.

Passive selection was performed using the E200, the E200-BSA conjugate,the E200, ovalbumin conjugate and the E200-DT conjugate peptides whilesolution screening used the biotinylated peptide then assayed usingstreptavidin. Both passive and solution selections of the numerous leaddabs worked well with specific binders demonstrating good sequencediversity in the form of the single V_(H) domains. Screening against theE200 peptide and smaller parts (200-208 and 207-215) revealed the leaddabs bound to different regions. Those with the best solutionproperties, being the highest monomer solubility were carried forward.Those demonstrating biphasic Biacore binding characteristics were notcarried forward. All showed uM binding to the E200 peptide. Ultimately atotal of five screening rounds were undertaken as shown in Example 5. Anexample of the results in Round 2 are shown in FIG. 1.

An example of dAb binding to cancer tissue follows in which humancervical cancer tissue was stained with c-Myc-labelled dAb PEP2-4 andthen developed using mouse anti-Myc antibody (1:600) followed by theBiocare Medical Mach4 secondary polymer detection system and DAB. Toinhibit binding, the peptide substrate was added to the primary atconcentrations of 0 (FIG. 2), 25 nM (no loss of binding), 0.25 uM (noloss of binding), 10 uM (no loss of binding), 0.1 mM (FIG. 3) and 1 mM(FIG. 4).

No inhibition of binding was observed at a concentration less than 0.01mM indicating the ideal for 50% inhibition is about 40-50 uM.

A second set of serial sections is shown in FIGS. 5-7 from differenttissue sections, magnification also 10×.

The difference between 0 and 10 uM added competing peptide in contrastwas minimal as shown in FIGS. 8 (no peptide) and 9 (10 uM peptide).

There is clear inhibition at 100 uM with no inhibition at 10 uMindicating that the binding at 50% inhibition appears to be about 40-50uM in this system.

A section of human melanoma tissue similarly stained with 20 nM PEP2-4dAb is shown in FIG. 10 below:

Conclusion:

Antigen binding sites in the form of dAb leads for high affinity P2X₇binding to PC3 cells were identified. Whether these antigen bindingsites interact with a linear or conformational epitope was unknown andsubsequently investigated. Refinement of the leads required addedscreening against a conformational epitope representing the shape of theE200 target antigen binding site as expressed on cancer cells

Example 2 Determining Activity of dAb Leads in dAb-Fc Format

Objective:

The experiments described here have been to improve affinity of antigenbinding sites that bind the E200 peptide through formatting lead dabs asdAb-Fc.

Background:

Co-operative binding of the lead dabs was achieved by producing standardformat dAb-Fc with human type IgG1 Fc subtype. These formats enabledmore considered screening of the lead dAb clones by enabling theelimination of high affinity lead dabs for which formatting as dAb-Fcprovided little benefit due to solubility issues. Favourableconformational solutions would then be selected for additional rounds ofscreening.

Results: The first formatted dabs PEP2-4 and PEP2-5 that had been chosenas high affinity leads from Example 1 showed little additional bindingto the E200 peptide whereas PEP2-2 and others (2-47, 2-42) benefitedwith a typical improvement in K_(D) of 100-1000 times. Formatting of thevarious leads resulted in good expression as revealed in the SDS-PAGEgel in FIG. 11.

The improvement in binding is evident with the leads including PEP2-2,PEP2-42, PEP2-47 shown in FIG. 12 in which the Biacore chip was coatedwith 100 RU of E200 and each dAb-Fc run at 100 nM.

Example 3 Determination of a Conformational Epitope for Screening dAbLeads Against

Objective:

The experiments described here have been to determine an appropriateconformational epitope for finding dAbs that bind the E200 peptide andalso bind a conformational epitope.

Background:

The high affinity binders are to bind to a non functional P2X₇ receptorextra cellular domain. The sequence of P2X₇ is shown in SEQ ID NO:1.There are a number of possible constructs that could be developed but wehad to determine which of these would model the conformational epitopesas observed on a live cancer cell. We particularly needed a target thatcould be bound to a solid phase for later affinity maturationexperiments.

We started with ECD1 that has the structure 47-332 because thisconstitutes all the amino acids forming the extracellular domain betweenthe transmembrane domains TM1 and TM2 including the putativeintramembraneous segment at the C-terminus of the segment from 325-332.By including all the residues it was considered likely that thestructure around the target E200 would be conserved.

Materials and Methods:

ECD1 was constructed recombinantly using standard molecular biologyprocedures and expressed in E. coli cells as soluble protein andformatted as ECD-Fc and in pDisplay for immunofluorescence, WesternBlotting and flow cytometry. The pDisplay structure had the form shownin the schematic in FIG. 13.

Results:

Cell surface expression of P2X₇ in the form of the wild type (WT) and intwo non-functional full length mutant forms (R307Q and E496A) werecompared along with the ECD1 in HEK293E cells and measured with WesternBlot. Cell lysates and cell surface expression was compared in all threeforms and the labelling to the ECD1 added. Anti-cadherin was used as astandardisation control. The cells were biotinylated withsulfo-NHS-SS-biotin, the reaction quenched and lysis performed with milddetergent. At this stage an aliquot was retained for indication of totalcell protein. Biotinylated protein was captured with neutravidin resinthat was washed and eluted with 50 mM DTT. The supernatant was retainedfor an indication of the intracellular pool of specific protein. Thesamples were then run on standard reducing SDS PAGE/Westerns (FIG. 14).

Cell surface expression indicates a reduction in the levels of thenon-functional mutants compared with WT on the cell surface. The ECD1expression from expressed pDisplay is efficiently high. This form of theprotein is labelled by antibodies to the non-functional form of thereceptor, the tumour specific form and can therefore be considered apossible tumour representative form. Monocytes, in contrast, expressingthe WT form, were unable to bind the dAbs. The efficiency of binding ofthe dabs to the pDisplayECD1 was lower than the levels of expressionindicated should have been the case. This indicates that the targetepitope is sterically hindered from binding on live cells and that thestructure of ECD1 is sub-optimal.

Conclusion:

While ECD1 construct was bound by dAb leads indicating binding to aconformational epitope, binding was suboptimal which raised thequestions concerning whether this construct would be useful for affinitymaturation studies.

Example 4 Determining a Further Construct for Affinity Maturation ofLead dAbs

Objective:

To produce a construct that could be used in affinity maturationstudies.

Background:

Example 3 revealed that certain ECD isoforms might not reproduceconformational epitopes of P2X₇ as observed on live tumours. We decidedto pursue a further construct in the form of the structure 47-306(ECD2).

Materials and Methods:

ECD2 was constructed recombinantly as in Example 3, in soluble form, Fcformat and as pDisplay for immunofluorescence, Western Blotting and flowcytometry.

Results:

ECD2 expression as an Fc construct is shown in FIG. 15. A reducingSDS-PAGE with Protein A fractions shown in two forms: WT (functional)and K193A (non functional) mutant forms. dAbs were identified that bindthe ECD2 construct. NB is an aliquot of the supernatant representingprotein not bound by Protein A.

The dAb-Fc species PEP2-4 and PEP2-5 along with control dAb HEL4 wererun on non-reduced and reduced gels and corresponding Westerns run onthe fractions revealed with anti-P2X₇ antibody (FIG. 16). Both dAb-Fcexpression and ECD2-Fc expression is clear. The reduced gels showspecific label on the ECD2Fc of the anti-P2X₇ antibody at 62 kDa with alower molecular weight proteolytic fragment (single chain) at 31 kDa.The corresponding Western shows reactivity with both ECD2 bands but nonewith HEL4Fc, PEP2-4Fc or PEP2-5Fc.

Binding by flow cytometry to live HEK293E cells expressing pDisplay-ECD2was clearly improved (FIG. 17). Gating live cell binding with HEL4 asthe control negative binder showed clear improvements with a higherpercentage of positive cells detected with lead dAbs indicating thetarget epitope was less sterically hindered and available for binding(FIG. 18).

Conclusion:

Antigen binding sites have been identified that bind the non-functionalP2X₇ receptor on live cells and ECD2. The removal of residues 307-332,commencing an estimated 3 nm from the E200 epitope site, has improvedbinding with the removal of partial steric hindrance. No loss of E200conformation occurs even though the segment 307-332 would be expected tostabilise the protein fold as it interacts closely with the N-terminalsegment.

Example 5 Generating Various High Affinity Binders

Objective: To generate antigen binding sites with high affinity for thenon-functional P2X₇ receptor.

Background:

The antigen binding sites from Example 1 having the following sequences(SEQ ID NOS 209-211, 7-8, 212, 49-50, 213, 46-47, 214, 4-5, 215, 16-17,216, 13-14, 217, 43-44, 218-219, 23, 220, 34-35, 221, 40-41, 222, 37-38and 223, respectively, in order of appearance:

CDR1          CDR2                  CDR3 WTSSYAMS---AISGSGGSTYYADSVKG---CAKSYGA--------FDY PEP2-2RNHD.G---AISGSGGS.........---..EPKPMDTE-----..Y PEP2-47PMKD.G---AISGSGGS.........---..EPSHFDRP-----..Y PEP2-42DNVE.S---SIGSKGED.........---..QTVNVPEPA----.AY PEP2-1DNEP.G---S.AD..NH.........---...QR.LNRYRAQ--..Y PEP2-5PASN..---S.TA..YR.........---...QGQISNFPR---..Y PEP2-4GM.N..---S.NAT..R.........---...FNRFSHRQYN--..Y PEP2-34......---T.TSD.LR.........---...VHTFANRSLN--..Y PEP2-7GA.S..---T.N...LA.........---...CSSCTSLNAN--..Y PEP2-11AR.P.A---S.D.G.LQ.........---...ASAPKYFR----..Y PEP2-30AK.P.V---S.GPG.AR.........---...PWRVYSYDR---..Y PEP2-13...A.A---T.D.N.LI.........---...LQRYDRYTLN..Ywere used as starting points for iterative rounds of randomization andscreening subject to issues of binding in the Fc format, solubility andpossession of a uniphasic dissociation trace on Biacore. PEP2-2 andPEP2-47 possessed the requisite characteristics and were selected foraffinity maturation even though they surprisingly had lower singledomain affinity for the ECD2 conformational and E200 peptide targetsthan other lead dabs such as PEP2-4 and PEP2-5.Materials and Methods:

The selected V_(H) domains including 2-2, 2-47 and daughters ereaffinity matured through 6 rounds of sequence diversification thatincluded all CDRs as well as all framework regions through NNSdiversification that sampled all 20 amino acids at each position. Thescaffold of the V_(H) library originated from the human V_(H) that gaverise to the HEL4 control non-binder and the diverse positive binders hasthe sequence:

(SEQ ID NO: 224) VHD EVQLLEPGGGLVQPGGSLRLSCAASGVNVSHDSMTWVRQAPGKGLEWVSAIRGPNGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASGARHADTERPPSQQTMPFWGQGTLVTVSS

Error-prone libraries were generated with a 2.7 amino acid error rate.Pools of clones were screened against the E200 initially and then theECD2 by phage ELISA for increased binding affinity. Eight error-pronelibraries were subcloned into the soluble dAb expression vector pDOM38without tag. Passive selection was carried out until Round 3. A total of1000 clones were screened by Biacore from Round 5 libraries PEP2-42,PEP2-pooled and the Round 4 library PEP2-pooled. The pool of clonesrepresents PEP2 clones 2-1, 2-2, 2-11, 2-13, 2-30, 2-34, 2-42 and 2-47.Improvement in off-rates by Biacore were observed. ELISA screeningagainst 1 nM biotinylated E200 showed EC₅₀ improvement from the range10⁷ to 10⁶ ug/mL in Round 3 to 10⁴ ug/mL in Round 5, well above controldAbs.

Biacore tracing of selected PEP2-42 clones to E200 peptide are shown inFIG. 19. The parent clone and HEL4 control dabs are at the bottom of thefigure. Sequence variations of the selected clones are shown in thefollowing figure. The 32 clones shown all have improved off-rates.Off-rate curves fell into two families and clones were chosenaccordingly (FIG. 20) with E/F (blue at left) representing a classicaloff-rate curve and G/H (red at left) an irregular biphasic type. K_(D)values are 76 nM for clone 6 and 200 nM for clone 7.

Determination of biochemical and/or biophysical characteristics of theantigen binding sites were obtained by SEC-MALLS. Those with monomericsolution characteristics were selected over those with a propensity toaggregate. Clones were generally found with a solubility in PBS>10mg/mL.

NNS screening, particularly of part of the variable CDR3 region, butextending to critical residues in F4 such as the residues 103-105 wasused to refine antigen binding.

Results:

The affinity maturation family tree of antibodies is shown in the FIG.21. An example of the improved binding by Biacore is shown in the formof the clone PEP2-2-3Fc in FIG. 22. The channel was coated with 10 RUE200 peptide and then loaded with 100 pM, 250 pM, 500 pM and 1 nMPEP2-2-3 in ascending order on the figure. Curve fitting reveals a K_(D)of 130 pM. The corresponding value for the unformatted dAb PEP2-2-3against E200 is 7 nM, showing a more moderate increase in binding forthe high affinity dabs when formatted as dAb-Fc compared with theincrease from the parent dabs such as PEP2-2 that increased from 1 uM to300 pM.

Corresponding values for the K_(D) when measured against ECD2 in eithersolution form or as a ECD-Fc construct showed significantly lowerbinding against the conformational epitope, with PEP2-2-3 Fc producing avalue of 1.5 nM, PEP2-2-1 560 pM and PEP2-472-1 584 pM as examples.

Examples of PEP2-Fc KD derived from Biacore using E200 are shown in thefollowing Table.

PEP-Fc K_(D) (pM) 2-2 300 2-2-2 100 2-2-3 130 2-2-1-1 90 2-42 5,5002-42-1 120 2-47 7500 2-47-1 110 2-247-1 190 (2-2/2-47-1 CDR crossover)2-247-2 450 (2-2-1/2-47-1 CDR crossover) 2-472-1 90 (2-47-1/2-2-2 CDRcrossover)

The effect of NNS screening on position 103 in PEP2-2-1 is shown in FIG.23. Trace 1 is buffer only and Trace 5 is a typical example of improvedbinding obtained by exchanging the Trp for an Arg residue.

Binding of selected lead clones to HEK293 cells expressing mock control(no binding), pDisplay-ECD1 (moderate binding), pDisplay-ECD2 (higherbinding) and pDisplay control (no binding) is seen in FIG. 24.

The lead clones bind specifically and competitively to the targetantigen and can be competed off with the addition of the soluble ECD2.As an example FIG. 25 shows PEP2-2-1 Fc at 50 nM is competed off with 1uM of soluble ECD2. An SA Biacore chip is coated with E200-biotinpeptide. Data shown is from 20 RU coated channel with a flow rate of 20uL/min in HBS-EP buffer. The HEL 4 Fc neither binds nor is affected bythe addition of the ECD2. Similar results are achieved in competing offthe PEP2-2-1 Fc with E200 at 5 uM or the ECD2 Fc construct at 1 uM.

Flow cytometry of binding of several lead dAb-Fc antigen binders to livecancer cells is shown in the following examples. These include: prostatePC3 (FIG. 26), breast MDA-MB 231 (FIG. 27), ovarian SKOV-3 (FIG. 28),Renal 786-0 (FIG. 29), Melanoma G361 (FIG. 30) and Lung NCI-H596 (FIG.31) cell lines.

Non-crossreactivity with functional P2X₇ receptors on lymphocytes andmonocytes was examined with flow cytometry. An example is shown in FIG.32 in which the two dAb Fc clones PEP2-2-1 and PEP2-2-3 Fc showed nobinding above the HEL4 Fc control background. In contrast, binding tolive cancer cells such as prostate LNCap is clear (green in FIG. 33,with the HEL4 control in red showing no binding above the secondary andthe HLA positive control shown in blue).

Direct cell killing or growth inhibition, as measured using the CellTiter Blue Assay, was monitored with the lead clones PEP2-2-1 andPEP2-2-3 using a variety of cell lines. Over a 3 or 5 day growth cycle,the control cells grew while the net growth in the presence of the 2-2-1Fc or 2-2-3 Fc was measured as a proportion of the growth in thepresence of the HEL4 Fc control. FIG. 34 shows PC3 cell growthprogressively inhibited as 2-2-1 or 2-2-3 are titrated up to 40 ug/mLover 5 days whereas the control cells are unaffected by HEL4 Fc. Thecolorectal cancer cell line COLO205 shows more sensitivity with both2-2-1 and 2-2-3 Fc causing significant growth inhibition at 3 days whileat 5 days, no cells remain even at 2.5 ug/mL (FIG. 35). Similarly themelanoma cell line A375 shows significant cell killing at 3 days whileat 5 days no cells remain (FIG. 36).

Conclusion:

Antigen binding sites that have high affinity for the non-functionalP2X₇ receptor on live cells were identified, sequenced and biophysicallycharacterised. Their effects on cell function were examined.

Example 6 Future Experiments

Objective:

To further enhance affinity of the lead dabs through additional targetedNNS screening of residues involved in direct binding to the antigen andin residues enabling the CDRs to pack more efficiently. To improvestability and solubility of antigen binding sites by modifying the Fc.To improve the efficiency of cell killing.

Materials and Methods:

Standard techniques to enhance binding affinity such as additionalrounds of NNS screening will be performed. The clones produced will bescreened by Biacore to find those with improved off rates and phageELISA against ECD2 (47-306). Additional screening using the CTB Assaywill be performed to identify clones with the most efficient combinationof binding affinity and killing capacity.

Expected Results:

Clones with at least one log lower binding constants are expected to beisolated that also kill cancer cells more efficiently than existingleads. As an example, new high affinity lead dAb domains (no Fc format)such as PEP2-2-12 in FIG. 36 show a KD against the ECD2 domain of 945 pMwhereas the parent PEP2-2-1 exhibits a KD of 560 pM as an Fc constructwith associated co-operative binding. The construction of leads withdifferent Fc domains will enable the influence of the Fc on solubilityproperties and cell killing to be examined. Examples are the addition ofmouse type IgG2a Fc in place of human IgG type 1 Fc.

The labelling of high affinity single domain species would enable themto be used for systemic screening purposes. An example is shown in FIG.38 in which an Alexa488 label has been attached to the dAb domainPEP2-2-12 and similar Biacore affinity determination suggests a K_(D) of174 pM. A high affinity lead with different parent is shown in FIG. 39where PEP2-472-12Alexa488 domain is measured with a KD of 156 pM.

The invention claimed is:
 1. An antigen binding site for binding to aP2X₇ receptor, the antigen binding site being defined by generalformula:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 wherein: FR1, FR2, FR3 and FR4 are eachframework regions; CDR1, CDR2 and CDR3 are each complementaritydetermining regions; wherein: CDR1 has a sequence: RNHDMG SEQ ID NO: 7;CDR2 has a sequence: AISGSGGSTYYANSVKG SEQ ID NO: 53; CDR3 has asequence: EPKPMDTEFDY SEQ ID NO:
 9. 2. An antigen binding site forbinding to a P2X₇ receptor according to claim 1, wherein FR4 has asequence: PSPGTLVTVLE SEQ ID NO: 169, WGQGTLVTVSS SEQ ID NO: 153,WGQGTLVTVLS SEQ ID NO: 154, RSPGTLVTVSS SEQ ID NO: 155, PSPGTQVTVSS SEQID NO: 156, PSPGTLVTVSS SEQ ID NO: 157, RSQGTLVTVSS SEQ ID NO: 158,WSQGTLVTVSS SEQ ID NO: 159, RGQGTLVTVSS SEQ ID NO: 160, RFQGTLVTVSS SEQID NO: 161, WSPGTLVTVSS SEQ ID NO: 162, GSPGTLVTVSS SEQ ID NO: 163,WGPGTLVTVSS SEQ ID NO: 164, RGPGTLVTVSS SEQ ID NO: 165, CGPGTLVTVSS SEQID NO: 166, RSCGTLVTVSS SEQ ID NO: 167, or RSPGTLVTVLE SEQ ID NO: 168.3. A nucleic acid encoding an antigen binding site according to claim 1.4. A method for the treatment of cancer that expresses non-functionalP2X₇ receptor in an individual including the step of administering anantigen binding site according to claim 1 to an individual requiringtreatment for said cancer.
 5. The nucleic acid of claim 3, wherein FR4has a sequence: PSPGTLVTVLE SEQ ID NO: 169, WGQGTLVTVSS SEQ ID NO: 153,WGQGTLVTVLS SEQ ID NO: 154, RSPGTLVTVSS SEQ ID NO: 155, PSPGTQVTVSS SEQID NO: 156, PSPGTLVTVSS SEQ ID NO: 157, RSQGTLVTVSS SEQ ID NO: 158,WSQGTLVTVSS SEQ ID NO: 159, RGQGTLVTVSS SEQ ID NO: 160, RFQGTLVTVSS SEQID NO: 161, WSPGTLVTVSS SEQ ID NO: 162, GSPGTLVTVSS SEQ ID NO: 163,WGPGTLVTVSS SEQ ID NO: 164, RGPGTLVTVSS SEQ ID NO: 165, CGPGTLVTVSS SEQID NO: 166, RSCGTLVTVSS SEQ ID NO: 167, or RSPGTLVTVLE SEQ ID NO: 168.6. The method of claim 4, wherein FR4 has a sequence: PSPGTLVTVLE SEQ IDNO: 169, WGQGTLVTVSS SEQ ID NO: 153, WGQGTLVTVLS SEQ ID NO: 154,RSPGTLVTVSS SEQ ID NO: 155, PSPGTQVTVSS SEQ ID NO: 156, PSPGTLVTVSS SEQID NO: 157, RSQGTLVTVSS SEQ ID NO: 158, WSQGTLVTVSS SEQ ID NO: 159,RGQGTLVTVSS SEQ ID NO: 160, RFQGTLVTVSS SEQ ID NO: 161, WSPGTLVTVSS SEQID NO: 162, GSPGTLVTVSS SEQ ID NO: 163, WGPGTLVTVSS SEQ ID NO: 164,RGPGTLVTVSS SEQ ID NO: 165, CGPGTLVTVSS SEQ ID NO: 166, RSCGTLVTVSS SEQID NO: 167, or RSPGTLVTVLE SEQ ID NO: 168.